U.S. patent application number 13/809475 was filed with the patent office on 2013-05-09 for anti-addl monoclonal antibody and uses thereof.
The applicant listed for this patent is Renee C. Gaspar, Alexander McCampbell, Paul J. Shughrue, Fubao Wang, Weirong Wang, Min Xu, Ningyan Zhang, Wei-Qin Zhao. Invention is credited to Renee C. Gaspar, Alexander McCampbell, Paul J. Shughrue, Fubao Wang, Weirong Wang, Min Xu, Ningyan Zhang, Wei-Qin Zhao.
Application Number | 20130115227 13/809475 |
Document ID | / |
Family ID | 45470057 |
Filed Date | 2013-05-09 |
United States Patent
Application |
20130115227 |
Kind Code |
A1 |
Gaspar; Renee C. ; et
al. |
May 9, 2013 |
ANTI-ADDL MONOCLONAL ANTIBODY AND USES THEREOF
Abstract
Disclosed are antibodies that bind amyloid beta-derived
diffusible ligands, also known as ADDLs. The antibodies are
selective for ADDLs, can penetrate the brain, and are useful in
methods of detecting ADDLs and diagnosing Alzheimer's disease. The
antibodies also block binding of ADDLs to neurons, assembly of
ADDLS, and tau phosphorylation and are there useful in methods for
the preventing and treating diseases associated with ADDLs.
Inventors: |
Gaspar; Renee C.;
(Souderton, PA) ; Shughrue; Paul J.; (West
Chester, PA) ; Wang; Fubao; (Dresher, PA) ;
Wang; Weirong; (Harleysville, PA) ; Zhang;
Ningyan; (Ambler, PA) ; Zhao; Wei-Qin; (North
Wales, PA) ; Xu; Min; (Ambler, PA) ;
McCampbell; Alexander; (Chalfont, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Gaspar; Renee C.
Shughrue; Paul J.
Wang; Fubao
Wang; Weirong
Zhang; Ningyan
Zhao; Wei-Qin
Xu; Min
McCampbell; Alexander |
Souderton
West Chester
Dresher
Harleysville
Ambler
North Wales
Ambler
Chalfont |
PA
PA
PA
PA
PA
PA
PA
PA |
US
US
US
US
US
US
US
US |
|
|
Family ID: |
45470057 |
Appl. No.: |
13/809475 |
Filed: |
July 13, 2011 |
PCT Filed: |
July 13, 2011 |
PCT NO: |
PCT/US2011/043866 |
371 Date: |
January 10, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61364210 |
Jul 14, 2010 |
|
|
|
Current U.S.
Class: |
424/172.1 ;
435/375; 435/7.1; 435/7.92; 506/9; 530/388.2; 530/389.1 |
Current CPC
Class: |
A61K 39/3955 20130101;
C07K 2317/64 20130101; A61P 43/00 20180101; A61K 2039/505 20130101;
C07K 2317/90 20130101; C07K 2317/92 20130101; C07K 2317/76
20130101; C07K 2317/55 20130101; A61P 25/28 20180101; C07K 2317/24
20130101; C07K 2317/565 20130101; C07K 16/18 20130101; A61K 45/06
20130101; C07K 2317/94 20130101 |
Class at
Publication: |
424/172.1 ;
530/389.1; 530/388.2; 435/375; 435/7.1; 506/9; 435/7.92 |
International
Class: |
C07K 16/18 20060101
C07K016/18 |
Claims
1. An isolated antibody, or an antigen binding fragment thereof,
that binds amyloid .beta.-derived diffusible ligands comprising:
(a) a light chain variable region comprising, (i) a CDR1 of SEQ ID
NO: 1, (ii) a CDR2 of SEQ ID NO: 2, and (iii) a CDR3 having the
sequence Phe-Gln-Gly-Ser-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5 (SEQ ID NO: 3),
wherein Xaa1 is Arg, Lys or Tyr, Xaa2 is Val, Ala, or Leu, Xaa3 is
Pro, His, or Gly, Xaa4 is Ala, Pro, or Val, and Xaa5 is Ser, Gly,
or Phe; and (b) a heavy chain variable region comprising, (i) a
CDR1 of SEQ ID NO: 4, (ii) a CDR2 of SEQ ID NO: 5, and (iii) a CDR3
of SEQ ID NO: 6.
2. The isolated antibody or antigen-binding fragment of claim 1
wherein the light chain variable region CDR3 is selected from the
group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ
ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13.
3. The isolated antibody or antigen-binding fragment of claim 1
wherein the light chain variable region CDR3 is SEQ ID NO: 10.
4. The isolated antibody of claim 1 wherein the light chain
variable region of said antibody comprises SEQ ID NO: 15 and the
heavy chain variable region of said antibody comprises SEQ ID NO:
17.
5. The isolated antibody of claim 1 further comprising a heavy
chain constant region of SEQ ID NO: 21.
6. The isolated antibody or antigen-binding fragment of claim 1
further comprising a light chain variable region CDR1 having the
sequence
Arg-Ser-Ser-Gln-Ser-Ile-Val-His-Ser-Xaa1-Gly-Xaa2-Thr-Thy-Leu-Glu
(SEQ ID NO: 53), wherein Xaa1 is Asn, Ser, Thr, Ala, Asp or Glu and
Xaa2 is Asn, His, Gln, Ser, Thr, Ala, or Asp.
7. The isolated antibody or antigen-binding fragment of claim 1
further comprising a light chain variable region CDR2 having the
sequence Lys-Ala-Ser-Xaa1-Arg-Phe-Ser (SEQ ID NO: 54), wherein Xaa1
is Asn, Gly, Ser, Thr, or Ala.
8. The isolated antibody of claim 1, wherein the antibody is a
monoclonal antibody.
9. A pharmaceutical composition comprising the antibody or antigen
binding fragment of claim 1 in admixture with a pharmaceutically
acceptable carrier.
10. A method for attenuating binding of amyloid .beta.-derived
diffusible ligands to a neuron comprising contacting the neuron
with the antibody or antigen binding fragment of claim 1 so that
binding of A.beta.-derived diffusible ligands to the neuron is
attenuated.
11. A method for inhibiting assembly of amyloid .beta.-derived
diffusible ligands comprising contacting a sample containing
amyloid .beta.1-42 peptides with the antibody or antigen binding
fragment of claim 1 thereby inhibiting assembly of A.beta.-derived
diffusible ligands.
12. A method for inhibiting the phosphorylation of tau protein at
Ser202/Thr205 comprising contacting a sample containing a tau
protein with the antibody or antigen binding fragment of claim 1
thereby inhibiting the phosphorylation of tau protein at
Ser202/Thr205.
13. A method for attenuating the symptoms of a disease associated
with amyloid .beta.-derived diffusible ligands comprising
administering an effective amount of the pharmaceutical composition
of claim 9.
14. A method for identifying a putative therapeutic agent that
attenuates the binding of amyloid .beta.-derived diffusible ligands
to neurons comprising (a) contacting a composition comprising a
neuron with amyloid .beta.-derived diffusible ligands in the
presence of an agent; (b) contacting the composition with the
antibody or antigen binding fragment of claim 1; and (c) detecting
the amount of antibody or antigen binding fragment bound in the
presence of the agent, wherein a decrease in the amount of antibody
or antigen binding fragment bound in the presence of the agent as
compared to the amount of antibody bound in the absence of the
agent indicates that the agent is a putative therapeutic agent for
attenuating binding of amyloid .beta.-derived diffusible ligands to
neurons.
15. A method for detecting amyloid .beta.-derived diffusible
ligands in a sample comprising contacting a sample with the
antibody or antigen binding fragment of claim 1 and determining the
presence of a complex comprising the amyloid .beta.-derived
diffusible ligands and said antibody or antigen binding
fragment.
16. A method for diagnosing a disease associated with amyloid
.beta.-derived diffusible ligands comprising contacting a sample
with the antibody or antigen binding fragment of claim 1 and
determining the presence of a complex comprising the amyloid
.beta.-derived diffusible ligands and said antibody or antigen
binding fragment, wherein the complex is diagnostic of a disease
associated with amyloid .beta.-derived diffusible ligands.
17. A kit for detecting amyloid .beta.-derived diffusible ligands
comprising the antibody or antigen binding fragment of claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 USC .sctn.119 to
U.S. Provisional Application No. 61/364,210, filed Jul. 14,
2011.
FIELD OF THE INVENTION
[0002] The present invention relates to monoclonal antibodies for
use in the treatment of Alzheimer's disease. The invention also
provides compositions comprising monoclonal antibodies and methods
of using the compositions as biomarkers or for diagnosing and
treating diseases associated with amyloid beta (A.beta.) and
A.beta.-derived diffusible ligands (ADDLs).
BACKGROUND OF THE INVENTION
[0003] Alzheimer's disease (AD) is characterized by the progressive
loss of cognitive function and the accumulation of amyloid beta
(A.beta.) plaques in regions associated with learning and memory.
While A.beta. plaques were once thought to play a central role in
the pathogenesis of AD, a growing body of evidence suggests that
the A.beta.-derived diffusible ligands (ADDLs) may be responsible
for the disease-associated neuronal dysfunction and cognitive
decline (Walsh and Selkoe, 2004, Protein Pept. Lett., 11: 213-228).
ADDLs are small, soluble oligomers of A that are abundant in AD,
but not normal, brains (McLean et al., 1999, Ann. Neurol., 46:
860-866; Gong et al., 2003, Proc. Natl. Acad. Sci. USA, 100:
10417-10422). In vitro studies have shown that ADDLs, isolated from
AD brain or synthetic preparations, bind to a subpopulation of
cortical and hippocampal neurons (Gong et al., 2003; Klein et al.,
2004, Neurobiol. Aging, 25: 569-580; Lacor et al., 2004, J.
Neurosci., 24: 10191-10200; Shughrue et al., 2010, Neurobiol.
Aging, 31: 189-202), while little or no binding was detected with
fibrillar or monomer A.beta. preparations (Lacor et al., 2004;
Hepler et al., 2006, Biochemistry, 45: 15157-15167). Furthermore,
ADDL binding to neurons can be attenuated with both polyclonal
(Gong et al., 2003) and monoclonal antibodies (Lee et al., 2006, J.
Biol. Chem., 281: 4292-4299; De Felice et al., 2007, Neurobiol.
Aging 29: 1334-1347; Shughrue et al., 2010) generated against
ADDLs.
[0004] In rodent models, the central administration of ADDLs
induces deficits in rodent long term potentiation (LTP) and memory
formation (Walsh et al., 2002, Nature, 416: 535-539; Cleary et al.,
2004, Nat. Neurosci., 8: 79-84; Klyubin et al., 2005, Nat. Med.,
11: 556-561). The effect of oligomers on LTP was attenuated when
ADDLs were co-administered with an anti-A.beta. antibody or
administered to animals that were vaccinated with the A peptide
(Rowan et al, 2004, Exp. Gerontol., 39: 1661-1667). In a transgenic
model of AD, such as transgenic mice that produce human amyloid
precursor protein (hAPP), age-associated cognitive deficits have
been observed with elevated ADDL levels (Westerman et al., 2002, J.
Neurosci., 22: 1858-1867; Ashe, 2005, Biochem. Soc. Trans., 33:
591-594; Lee et al., 2006; Lesne et al., 2006, Nature, 440:
352-357). When hAAP mice were treated with an anti-ADDL antibody, a
significant improvement in cognitive performance was observed
without a concomitant decrease in A plaque load (Lee et al., 2006).
Together these findings suggest that ADDLs, and not A.beta.
plaques, are primarily responsible for cognitive impairment and
that the use of anti-ADDL antibodies may prove efficacious in the
treatment of AD. See also, US2006/0228349; U.S. Pat. No. 7,731,962,
WO 2007/050359; US2007/0218499, WO 2006/014478; U.S. Pat. No.
7,700,099; US 2008/01758835, WO 2006/055178.
[0005] Accordingly, there is a need for ADDL-selective therapeutic
antibodies for the prevention and treatment of AD. The present
invention meets this need.
SUMMARY OF THE INVENTION
[0006] The present invention is directed to an isolated antibody,
or fragment thereof, capable of differentially recognizing a
multi-dimensional conformation of one or more amyloid-.beta.
derived diffusible ligands (ADDLs) for the treatment of diseases
associated with ADDLs, such as Alzheimer's disease (AD). The
present invention also provides pharmaceutical compositions
comprising the isolated antibody of the invention, either alone or
in combination, with one or more therapeutically active agents,
carriers, or diluents.
[0007] The present invention is also directed to methods of use for
the isolated antibody, such as, methods for detecting ADDLs in a
sample, for inhibiting assembly of ADDLs, for identifying
therapeutic agents that prevents binding of ADDLs to neurons, and
for attenuating the symptoms of a disease associated with ADDLs,
and as a biomarker for use in the diagnosis of a disease associated
with ADDLs or for the detection of ADDLs in a sample.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 is a graphic representation of the ELISA binding of a
panel of humanized (h3B3) and affinity matured anti-ADDL (14.2,
7.2, 11.4, 9.2, 13.1, 17.1, and 19.3) antibodies and three
comparator antibodies (Camp 1, 2, and 3) to monomer A.beta., ADDLs
and fibrillar A.beta.. The background of this assay was determined
by removing the capture antibody from the ELISA (no mAb). Error
bars represent standard error of the mean.
[0009] FIG. 2 is a graphic representation of the ELISA binding of
anti-ADDL antibody 19.3 and antibody 3B3 to ADDLs or monomer
A.beta. (A.beta..sub.1-40) evaluated with an 11 point titration
curve.
[0010] FIG. 3 is a graphic representation of the ability of
anti-ADDL antibody 19.3 and 3B3 to block ADDL binding to primary
hippocampal neuronal cells after pre-incubation with increasing
concentration of the antibody. The ability of anti-ADDL antibody
19.3 to block ADDL binding to neurons was attenuated after heat
denaturing of the antibody. Error bars represent standard error of
the mean.
[0011] FIGS. 4A-4C are graphic representations of the ELISA binding
to ADDLs of the anti-ADDL antibody 19.3 (designated as WT in FIG.
4A) and two 19.3-derived anti-ADDL antibodies (FIGS. 4B and 4C)
after incubation up to one month at varying temperatures to
evaluate antibody stability. The 19.3-derived anti-ADDL antibodies
comprised a single amino-acid substitution of Asn33 within light
chain CDR1 to either Ser33 (19.3S33) or Thr33 (19.3T33) (SEQ ID
NOS: 55 and 56, respectively). Substitution of Asn33 with either
S33 (FIG. 4B) or T33 (FIG. 4C) resulted in improved antibody
stability versus the parental 19.3 antibody.
[0012] FIG. 5 is a graphic representation of the binding and
dissociation of anti-ADDL antibodies to immobilized human FcRn when
assessed with Biacore.TM. (GE Healthcare, Piscataway, N.J.). The
adjusted sensorgram shows initial binding at pH 6.0 and then the
dissociation of antibodies at pH 7.3 from 180 seconds. A report
point (Stability) was inserted at 5 seconds after the end of pH 6.0
binding and the "% bound" was calculated as
RU.sub.Stability/RU.sub.Binding (%).
[0013] FIG. 6A shows the alignment of the heavy and light chain
variable regions for anti-ADDL antibody 19.3 with a human germ line
with the complementary determining regions (CDRs) indicated in bold
type face. FIG. 6B is a three dimensional model of antibody 19.3
heavy and light variable regions showing the location of the
CDRs.
[0014] FIG. 7 is a graphical representation of the pharmacokinetic
(PK) profile of anti-ADDL antibodies 19.3 and 3B3 evaluated in
heterozygous 276 human FcRn mice (Jackson Laboratory (Bar Harbor,
Me.) following a single 10 mg/kg intravenous (IV) administration.
The concentration of antibody was measured at various time
intervals to determine the half-life (t.sub.1/2) of free anti-body
(19.3: 77.+-.6 hours; 3B3 respectively: 29.+-.9 hours).
[0015] FIG. 8 is a graphical representation of the PK of anti-ADDL
antibody 19.3 (in serum) assessed in six rhesus monkeys following
administration of a bolus intravenous (IV) or subcutaneous (SC)
dose of 5 mg/kg. A half-life (t.sub.1/2) of 254.+-.28 (274.+-.9)
hours was determined after IV administration and 204.+-.49
(219.+-.52) hours after SC dosing.
[0016] FIG. 9 is a graphical representation of the PK of anti-ADDL
antibody 19.3 assessed in primate (three male rhesus monkeys)
cerebrospinal fluid (CSF) using a cisterna magna ported rhesus
model following administration of a bolus IV dose of 5 mg/kg. At
about 48 hours post dose, the anti-ADDL antibody 19.3 was present
in the CSF at 0.1% of the concentration in serum.
[0017] FIGS. 10A-10D are representations of the ability of
anti-ADDL antibody 19.3, versus two comparator antibodies (Comp 1
and Comp2), to cross the blood-brain-barrier in a transgenic mouse
model that over-expresses human amyloid precursor protein (hAAP).
Mice were injected intravenously (IV) with .sup.125I-labeled
anti-ADDL antibody 19.3, or a comparator antibody, and the blood,
CSF and brain samples were collected two hours post-dose. Upon
assessment of the radioactivity distribution, 0.02% of anti-ADDL
antibody 19.3 was present in the CSF (FIG. 10A), while 0.19% was
seen in the brain (FIG. 10B). Similar levels were seen with the two
comparator antibodies. Immunocytochemical analysis demonstrated
localization of anti-ADDL antibody 19.3 (FIG. 10C, arrows) and a
concentration of anti-ADDL antibody 19.3 was visible with plaques
(FIG. 10D). The anti-ADDL antibody19.3 was able to penetrate into
the brain and bind ADDLs.
[0018] FIGS. 11A-11C are representations of the ability of
anti-ADDL antibody 19.3 to block the deposition of ADDLs into
growing plaques in a transgenic mouse model that over-expresses
hAAP. Biotinylated ADDLs (bADDLs) infused into the hippocampus of
12-month-old mice for four weeks (one injection per week) (FIG.
11A) labeled existing plaques (vehicle alone: FIG. 11B; antibody
19.3: FIG. 11C, ring). Immunocytochemical analysis was used to
assess the deposition of new material (ADDLs) (FIGS. 11B and
11C).
DETAILED DESCRIPTION OF THE INVENTION
[0019] The present invention is directed to antibodies, or an
antigen binding fragment, that bind amyloid .beta.
(A.beta.)-derived diffusible ligands (ADDLs), i.e. anti-ADDL
antibodies, and attenuate ADDL binding to neurons. Results from a
quantitative cell-based assay revealed that anti-ADDL antibodies
preferentially bound ADDLs, abated the binding of ADDLs to
hippocampal neurons, crossed the blood-brain barrier, and had an
improved pharmacokinetic (PK) profile.
[0020] In one embodiment the present invention is directed to an
isolated antibody, or an antigen binding fragment thereof; that
binds amyloid .beta.-derived diffusible ligands (ADDLs)
comprising:
[0021] (a) a light chain variable region comprising,
[0022] (i) a CDR1 having the sequence
Arg-Ser-Ser-Gln-Ser-Ile-Val-His-Ser-Asn-Gly-Asn-Thr-Tyr-Leu-Glu
(SEQ ID NO: 1),
[0023] (ii) a CDR2 having the sequence Lys-Ala-Ser-Asn-Arg-Phe-Ser
(SEQ ID NO: 2), and
[0024] (iii) a CDR3 having the sequence
Phe-Gln-Gly-Ser-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5 (SEQ ID NO: 3), wherein
Xaa1 is Arg, Lys or Tyr, Xaa2 is Val, Ala, or Leu, Xaa3 is Pro,
His, or Gly, Xaa4 is Ala, Pro, or Val, and Xaa5 is Ser, Gly, or
Phe; and
[0025] (b) a heavy chain variable region comprising,
[0026] (i) a CDR1 having the sequence
Gly-Phe-Thr-Phe-Ser-Ser-Phe-Gly-Met-His (SEQ ID NO: 4),
[0027] (ii) a CDR2 having the sequence
Tyr-Ile-Ser-Arg-Gly-Ser-Ser-Thr-Ile-Tyr-Tyr-Ala-Asp-Thr-Val-Lys-Gly
(SEQ ID NO: 5), and
[0028] (iii) a CDR3 having the sequence
Gly-Ile-Thr-Thr-Ala-Leu-Asp-Tyr (SEQ ID NO: 6).
[0029] In another embodiment the present invention is directed to
an isolated antibody, or an antigen binding fragment thereof, that
binds amyloid .beta.-derived diffusible ligands (ADDLs)
comprising:
[0030] (a) a light chain variable region comprising,
[0031] (i) a CDR1 having the sequence
Arg-Ser-Ser-Gln-Ser-Ile-Val-His-Ser-Xaa1-Gly-Xaa2-Thr-Tyr-Leu-Glu
(SEQ ID NO: 53), wherein Xaa1 is Asn, Ser, Thr, Ala, Asp or Glu and
Xaa2 is Asn, His, Gln, Ser, Thr, Ala, or Asp;
[0032] (ii) a CDR2 having the sequence Lys-Ala-Ser-Xaa1-Arg-Phe-Ser
(SEQ ID NO: 54), wherein Xaa1 is Asn, Gln, Ser, Thr, or Ala,
and
[0033] (iii) a CDR3 having the sequence
Phe-Gln-Gly-Ser-Arg-Leu-Gly-Pro-Ser (SEQ ID NO: 10); and
[0034] (b) a heavy chain variable region comprising,
[0035] (i) a CDR1 having the sequence
Gly-Phe-Thr-Phe-Ser-Ser-Phe-Gly-Met-His (SEQ ID NO: 4),
[0036] (ii) a CDR2 having the sequence
Tyr-Ile-Ser-Arg-Gly-Ser-Ser-Thr-Ile-Tyr-Tyr-Ala-Asp-Thr-Val-Lys-Gly
(SEQ ID NO: 5), and
[0037] (iii) a CDR3 having the sequence
Gly-Ile-Thr-Thr-Ala-Leu-Asp-Tyr (SEQ ID NO: 6).
[0038] In another embodiment the present invention is an isolated
antibody that binds ADDLs, i.e. an anti-ADDL antibody, or an
antigen binding fragment thereof, having a light chain variable
region CDR3 that is selected from the group consisting of 17.1,
having the sequence Phe-Gln-Gly-Ser-Arg-Val-Pro-Ala-Ser (SEQ ID NO:
7), 14.2, having the sequence Phe-Gln-Gly-Ser-Arg-Val-Pro-Pro-Gly
(SEQ ID NO: 8), 13.1, having the sequence
Phe-Gln-Gly-Ser-Lys-Ala-His-Pro-Ser (SEQ ID NO: 9), 19.3, having
the sequence Phe-Gln-Gly-Ser-Arg-Leu-Gly-Pro-Ser (SEQ ID NO: 10),
7.2, having the sequence Phe-Gln-Gly-Ser-Tyr-Ala-Pro-Pro-Gly (SEQ
ID NO: 11), 9.2, having the sequence
Phe-Gln-Gly-Ser-Arg-Ala-Pro-Pro-Phe (SEQ ID NO: 12), and 11.4,
having the sequence Phe-Gln-Gly-Ser-Arg-Val-Pro-Val-Arg (SEQ ID NO:
13). In a sub-embodiment the light chain variable region CDR3 is
SEQ ID NO: 10.
[0039] In still another embodiment of the present invention the
isolated anti-ADDL antibody further comprises a light chain
variable region of SEQ ID NO: 15 and a heavy chain variable region
of SEQ ID NO: 17.
[0040] In yet another embodiment of the present invention the
isolated anti-ADDL antibody further comprises a heavy chain
constant region of SEQ ID NO: 21.
[0041] In another embodiment of the present invention the isolated
anti-ADDL antibody is a monoclonal antibody.
[0042] Another embodiment of the present invention is a
pharmaceutical composition comprising an isolated anti-ADDL
antibody, or an antigen binding fragment thereof, in admixture with
a pharmaceutically acceptable carrier.
[0043] Another embodiment of the present invention is a method for
attenuating binding of ADDLs to a neuron comprising contacting the
neuron with an isolated anti-ADDL antibody, or an antigen binding
fragment thereof, so that binding of A.beta.-derived diffusible
ligands to the neuron is attenuated.
[0044] Another embodiment of the present invention is a method for
inhibiting the assembly of ADDLs comprising contacting a sample
containing amyloid .beta.1-42 peptides with an isolated anti-ADDL
antibody, or antigen binding fragment thereof, thereby inhibiting
the assembly of ADDLs.
[0045] Another embodiment of the present invention is a method for
inhibiting the phosphorylation of tau protein at Ser202/Thr205
comprising contacting a sample containing a tau protein with an
isolated anti-ADDL antibody, or an antigen binding fragment
thereof, thereby inhibiting the phosphorylation of tau protein at
Ser202/Thr205.
[0046] Another embodiment of the present invention is a method for
attenuating the symptoms of a disease associated with ADDLs
comprising administering an effective amount to a patient in need
thereof of the pharmaceutical composition comprising an isolated
anti-ADDL antibody, or an antigen binding fragment thereof.
[0047] Another embodiment of the present invention is a method for
identifying a putative therapeutic agent that attenuates the
binding of amyloid .beta.-derived diffusible ligands (ADDLs) to
neurons comprising:
[0048] (a) contacting a composition comprising a neuron with ADDLs
in the presence of an agent;
[0049] (b) contacting the composition with the isolated anti-ADDL
antibody, or an antigen binding fragment thereof; and
[0050] (c) detecting the amount of antibody or antigen binding
fragment bound in the presence of the agent,
[0051] wherein a decrease in the amount of antibody or antigen
binding fragment bound in the presence of the agent as compared to
the amount of antibody bound in the absence of the agent indicates
that the agent is a putative therapeutic agent for attenuating
binding of ADDLs to neurons.
[0052] Another embodiment of the present invention is a method for
detecting ADDLs in a sample comprising contacting a sample with an
isolated anti-ADDL antibody, or an antigen binding fragment
thereof, and determining the presence of a complex comprising the
ADDLs and said antibody or antigen binding fragment.
[0053] Another embodiment of the present invention is a method for
diagnosing a disease associated with ADDLs comprising contacting a
sample with an isolated anti-ADDL antibody, or an antigen binding
fragment thereof, and determining the presence of a complex
comprising the ADDLs and said isolated antibody or antigen binding
fragment, wherein the presence of said complex is diagnostic of a
disease associated with ADDLs.
[0054] Still another embodiment of the present invention is a kit
for detecting ADDLs comprising an isolated anti-ADDL antibody, or
an antigen binding fragment thereof, that binds ADDLs.
[0055] Monoclonal antibodies, which differentially recognize
multi-dimensional conformations of A.beta.-derived diffusible
ligands (ADDLs) are known in the art (see, U.S. Pat. No. 7,780,963,
U.S. Pat. No. 7,731,962, and U.S. Pat. No. 7,811,563, all of which
are incorporated herein by reference in their entirety), and have
been shown to reduce ADDL binding to neurons in cell based assays.
Anti-ADDL antibodies can distinguish between Alzheimer's disease
(AD) and control human brain extracts, can identify endogenous
oligomers in AD brain slices and on hippocampal cells, and can
neutralize endogenous and synthetic ADDLs in solution. Anti-ADDL
antibodies specifically bind one or more multi-dimensional
conformations of ADDLs, bind particular ADDLs derived from the
oligomerization of A.beta.42, while having reduced affinity for
other A.beta. peptides, including A.beta.1-40.
[0056] The present invention is directed to anti-ADDL antibodies,
specifically antibodies 17.1, 14.2, 13.1, 19.3, 19.3T33, 19.3S33,
7.2, 9.2, and 11.4, that preferentially bind ADDLs and that have
been characterized as to their specificity and selectivity for
ADDLs. Importantly, the specificity and selectivity of these
anti-ADDL antibodies of the present invention was not predictable
from the linear epitope of A.beta. to which they bound, nor was
this activity predictable from their ability to detect ADDLs by
Western blot, or from their ability to detect immuno-stained ADDLs
bound to neurons. Moreover, the differential ability of the
anti-ADDL antibodies of the present invention to neutralize ADDLs
and block binding to primary hippocampal neurons supports the
belief that anti-ADDL antibodies act through binding to a more
relevant, conformational epitope, which prevents ADDL binding to
neurons. One embodiment of the present invention, anti-ADDL
antibody 19.3, not only blocked the binding of ADDLs to primary
neurons, but also abated ADDL-induced changes to hippocampal spine
morphology, an indication that the impedance of ADDL-neural binding
has significant physiological ramifications, for example, neuronal
survival, neuronal connectivity and signal transduction. Anti-ADDL
antibody 19.3 also had an improved pharmacokinetic (PK) profile, as
compared with a previously known anti-ADDL antibody, 3B3, when
assessed in both in vitro and in vivo models. In addition, when
administered to transgenic mice that over-express a human form of
amyloid precursor protein (hAAP), anti-ADDL antibody 19.3 was shown
to penetrate the blood-brain-barrier and concentrate in the brain.
Since ADDLs are localized in the brain and act there to adversely
affect neuronal function, one of skill in the art would appreciate
and recognize that the penetration and concentration of antibody in
the brain would be beneficial for immunotherapy. Taken together,
these data demonstrate that selective anti-ADDL antibodies, such as
antibody 19.3, can block the binding of ADDLs to hippocampal
neurons, which are critically involved in learning and memory.
[0057] The utility of anti-ADDL antibodies for the treatment of AD
is based on a growing body of evidence that suggests that ADDLs,
and not amyloid plaques per se, play a fundamental role in the
cognitive decline associated with this disease (Walsh and Selkoe,
2004, Protein Pept. Lett., 11: 213-228). ADDLs are elevated in the
AD brain and induce deficits in behavioral and electrophysiological
endpoints when centrally administered to rodents (Walsh, et al.,
2002, Nature, 416: 535-539; Cleary, et al., 2004, Nat. Neurosci.,
8: 79-84; Klyubin, et al., 2005, Nat. Med., 11: 556-561; Balducci,
et al., 2010, Proc. Natl. Acad. Sci. USA, 107: 2295-2300). Deficits
in learning and memory have also been observed in a hAAP expressing
mouse model, with the onset of impairment associated with elevated
ADDL levels (Westerman, et al., 2002, J. Neurosci., 22: 1858-1867;
Ashe, 2005, Biochem. Soc. Trans., 33: 591-594; Lee, et al., 2005,
J. Biol. Chem., 281: 4292-4299; Lesne, et al., 2006, Nature, 440:
352-357). While the cellular and sub-cellular events that mediate
these effects on cognition are not fully understood, it is clear
that ADDLs bind to the synaptic terminals localized on the
dendritic processes of hippocampal neurons (Lacore, et al., 2004,
J. Neurosci., 24: 10191-1022) and alter the morphology and number
of dendritic spines (Lacor et al., 2007, J. Neurosci., 27: 796-807;
Shankar, et al., 2007, J. Neurosci., 27: 2866-2875; Shughrue, et
al., 2010, Neurobiol. Aging, 31: 189-202). The finding that ADDLs
bind to both GABAergic and glutamate neurons in the hippocampus
(Shughrue, et al., 2010), neurons critically involved in learning
and memory, which results in the internalization of AMPA receptors
(Zhao, et al., 2010, J. Biol. Chem., 285: 7619-7632) further
supports the belief that ADDLs directly or indirectly modulate
these neurotransmitter systems (see, for example, Venkitaramani, et
al., 2007, J. Neurosci., 27: 11832-11837).
[0058] In the present invention, a panel of anti-ADDL antibodies
derived from anti-ADDL antibody, 3B3 (U.S. Pat. No. 7,780,963 and
U.S. Pat. No. 7,811,563, which are hereby incorporated by reference
in their entirety), were assessed for their ability to block ADDL
binding to primary hippocampal neurons. Selected monoclonal
antibodies were then humanized and affinity matured for further
characterization. Lead antibodies, selected for their ability to
bind to ADDLs, were further assessed at a single concentration
using a three-pronged ELISA to determine antibody binding to
monomer A.beta., ADDLs, and fibrillar A.beta.. As shown in FIG. 1,
six of the seven affinity matured anti-ADDL antibodies,
specifically antibodies 14.2, 7.2, 11.4, 13.1, 17.1, and 19.3 were
ADDL preferring, when compared with monomer A.beta. and fibrillar
A.beta.. Subsequently an eleven point titration curve and ELISA
were used to ascertain the binding affinity of anti-ADDL antibodies
to ADDLs and monomer A.beta. (A.beta..sub.1-40) over a broad range
of concentrations. As shown in FIG. 2, the anti-ADDL antibodies 3B3
and 19.3 were highly ADDL selective. In addition, antibodies were
compared in a cell-based binding assay to determine the ability of
antibodies to block ADDL binding to neurons. As shown in FIG. 3,
ADDLs, pre-incubated with increasing concentrations of anti-ADDL
antibodies 3B3 and 19.3, were added to primary hippocampal neurons,
and a titration curve was used to show quantitatively the ability
of the antibody to block ADDL binding to neurons. Taken together,
these results show that anti-ADDL antibodies profoundly attenuate
neuronal binding in a cell-based format.
[0059] An assessment of the amino acid sequence was conducted to
identify potential sites of deamidation. Asparagine and aspartic
acid residues present in the CDRs of therapeutic antibodies are
known to undergo deamidation and isoaspartate formation (Valsak and
Ionescu, 2008, Curr. Pharm. Biotech., 9:468-481; Aswad et al.,
2000, J. Pharm. Biomed. Anal., 21:1129-1136), the formation of
which can alter the binding potency of an antibody and, in turn,
reduce antibody effectiveness for use as a therapeutic. Thus, those
of skill in the art would recognize and appreciate that the
presence of an asparagine or an aspartic acid within the CDRs for
the 19.3 antibody would not be desirable. Accordingly, Applicants
altered the asparagine residue at position 33 of the light chain
CDR1 to optimize the stability of the anti-ADDL antibody 19.3
(Table 4B). Derivatives of the 19.3 antibody were produced with the
substitution of serine (SEQ ID NO: 55), threonine (SEQ ID NO: 56),
or glutamic acid (SEQ ID NO: 67) for the asparagine at position 33
(SEQ ID NO: 1) in CDR1. The substitution of aspartic acid (SEQ ID
NO: 68) for the asparagine as position 33 was also generated as a
control. These changes will remove the possibility of deamidation
of asparagine at position 33 in CDR1. The 19.3 derivatives were
generated as described in Example 3 and characterized as described
in Example 4 as to derivatives with the serine (SEQ ID NO: 55),
threonine (SEQ ID NO: 56), glutamic acid (SEQ ID NO: 67), and
aspartic acid (SEQ ID NO: 68) substitutions, to evaluate the
stability of the new constructs. As shown in FIGS. 4B and 4C,
respectively, two representative derivatives, 19.3S33 (SEQ ID NO:
55) and 19.3T33 (SEQ ID NO: 56), had enhanced binding stability
following a one-month incubation at varying temperatures. Other
amino acid substitutions in the light chain CDR1 for the asparagine
at positions 33 and 35 (SEQ ID NO: 53) and in the light chain CDR2
for the asparagine at position 58 position (SEQ ID NO: 54) are
proposed in Tables 4B and 4C for further evaluation.
[0060] To determine the pharmacokinetics of the affinity matured
anti-ADDL antibodies of the present invention, a series of in vitro
and in vivo studies were conducted. The binding of antibodies to
the FcRn receptor at pH 6.0 has been shown to be predictive of
antibody half-life in humans (Zalevsky, et al., 2010, Nat.
Biotech., 28(2): 157-159) and at pH 7.3 (U.S. Ser. No. 61/307,182)
The binding and dissociation of the anti-ADDL antibodies of the
present invention to immobilized human FcRn was assessed with a
label free interaction analysis, such as that offered by
Biacore.TM. Life Sciences, Biacore.TM. T-100 (GE Healthcare,
Piscataway, N.J.). An adjusted sensorgram is used to show the
initial binding at pH 6.0 and then the dissociation of antibodies
at pH 7.3 from 180 seconds. A report point (Stability) was inserted
at 5 seconds after the end of pH 6.0 binding and the "% bound" was
calculated as RU.sub.Stability/RU.sub.Binding (%). As shown in FIG.
5, the off-rate for humanized 3B3 was markedly slower than the
seven anti-ADDL antibodies of the present invention, which included
antibody 19.3, and three comparator antibodies. In that a slow
off-rate is thought to be an indicator of poor in vivo PK, an
additional in vivo study was conducted in transgenic FcRn mice
(heterozygous 276 human FcRn mice, Jackson Laboratories, Bar
Harbor, Me.). When the transgenic FcRn mice were given 10 mg/kg
intravenously (IV) of either anti-ADDL antibody 3B3 or 19.3, a
significant difference in pharmacokinetics was determined. As shown
in FIG. 7, the half-life (t.sub.1/2) of anti-ADDL antibody 3B3 was
relatively short (29.+-.9 hours), which was consistent with the
prediction from the in vitro Biacore.TM. data, while the half-life
for anti-ADDL antibody 19.3 was significantly longer (77.+-.6
hours). Generally, poor PK, as seen with antibody 3B3, would
preclude further development of an antibody for use as a
therapeutic due to its short bioavailability.
[0061] To confirm the predicted half-life of anti-ADDL antibody
19.3 in primates, a primate pharmacokinetics study was conducted
for the antibody in a cohort of cisterna magna ported rhesus
monkeys. The animals were dosed with a single intravenous (IV)
bolus or subcutaneous (SC) injection of anti-ADDL antibody 19.3 (5
mg/kg) and blood samples collected after antibody administration.
Concurrently, CSF samples were collected from the cisterna magna
port at timed intervals and the concentration of anti-ADDL antibody
19.3 in serum and CSF was determined with an anti-human IgG ELISA
assay. When the animals were administered anti-ADDL antibody 19.3
by a single IV bolus injection a t.sub.1/2 of 254.+-.28 hours (FIG.
8) was observed, while a t.sub.1/2 of 204.+-.49 hours was observed
for the subcutaneous administration. In addition, Applicants found
that anti-ADDL antibody 19.3 was able to cross into the primate
CSF, where it increased in concentration during the first 48 hours
and peaked at about 0.1% of the antibody dosed (FIG. 9).
[0062] In an attempt to ascertain the quantity of antibody that
penetrates the blood-brain-barrier and enters the CSF and brain,
anti-ADDL antibody 19.3 and two comparator antibodies (Comp 1 and
Comp 2) were .sup.125I-labeled and administered to aged
(twelve-month old) mice that over-express hAAP, a rodent model for
AD. Two hours after IV dosing about 0.02% of antibody 19.3 was seen
in the CSF (FIG. 10A), while about 0.19% of antibody 19.3 was seen
in the brain (FIG. 10B). Similar levels were seen for the two
comparator antibodies (FIGS. 10A and 10B). When immunocytochemical
analysis was carried out on brain sections of the dosed mice and
the localization of anti-ADDL antibody 19.3 was determined (arrow
in FIG. 10C), a concentration of the antibody associated with the
deposition of A.beta. into plaques was observed (FIG. 10D). This
demonstrated that the anti-ADDL antibody 19.3 penetrated into the
CSF and was concentrated in the brain. Recently it was shown that
exogenous ADDLs were deposited into plaques when administered to
mice that over express hAAP (Gaspar, et al., 2010, Exp. Neurol.,
223: 394-400). Thus, the findings herein confirmed that the
localized anti-ADDL antibody19.3 bound to circulating ADDLs
associated with plaques.
[0063] To further evaluate the in vivo efficacy of anti-ADDL
antibodies, the ability of antibody 19.3 to block the deposition of
ADDLS into growing plaques was assessed in hAAP transgenic mice
following four weekly infusions of biotinylated ADDLs (bADDLs) into
the hippocampus of 12-month old mice to label existing plaques
(FIG. 11A). The animals then received four weekly intravenous
infusions of antibody 19.3 (FIG. 11A). The deposition of new
material (ADDLs) into growing plaques was assessed by
immunocytochemical analysis. As seen in FIGS. 11B and 11C,
anti-ADDL antibody 19.3 significantly reduced the deposition of
ADDLs into the periphery of existing plaques (FIG. 11C) as compared
to mice treated with vehicle alone (FIG. 11B). Taken together,
these results demonstrated that an anti-ADDL antibody, specifically
the 19.3 antibody, was able to cross the blood-brain-barrier, bind
ADDLs, and block the deposition of new material into growing
plaques.
[0064] ADDL binding may also have long-term effects on neurons.
Recent studies have shown that ADDL binding to hippocampal neurons
can initiate a signaling cascade that results in the
phosphorylation of tau (De Felice, et al., 2006, Neurobiol. Aging,
29: 394-400). One component of this signaling cascade, GSK-3.beta.,
has also been shown to be modulated by ADDL binding in vivo and in
vitro (Ma, et al., 2006, J. Neurosci. Res., 83: 374-384). Ma, et
al., 2006, found that passive immunization of hAAP mice with an
antibody that reduced ADDLs, also reduced GSK-3.beta. levels and
phosphorylation of tau in the cortex. This finding supports a link
between A.beta. and phosphorylated tau and suggests that ADDL
binding may trigger events that lead to the intracellular
aggregation of tau. Further, the data suggests that antibodies that
prevent the binding of ADDLs to neurons and the associated loss of
synaptic spines, such as the antibodies of the present invention
could ameliorate the cognitive and/or pathological outcomes
associated with Alzheimer's disease and related diseases.
[0065] Monoclonal antibodies, which differentially recognize
multi-dimensional conformations of A.beta.-derived diffusible
ligands, i.e., ADDLs, have now been generated. These antibodies
were humanized and, in some embodiments, affinity-matured. The
antibodies advantageously distinguish between Alzheimer's disease
and control human brain extracts, and identify endogenous oligomers
in Alzheimer's disease brain slices and in cultured hippocampal
cells. Further, the antibodies of the present invention neutralize
endogenous and synthetic ADDLs in solution. So-called "synthetic"
ADDLs are produced in vitro by mixing purified A.beta..sub.1-42
under conditions that generate ADDLs. See, U.S. Pat. No. 6,218,506.
The antibodies disclosed herein exhibit a high degree of
selectivity for ADDLs, with minimal detection of monomer A.beta.
species. Moreover, these antibodies differentially block the
ability of ADDL-containing preparations to bind primary cultures of
rat hippocampal neurons and immortalized neuroblastoma cell lines,
and also block ADDL assembly. This finding demonstrates that these
antibodies possess a differential ability to recognize a
multi-dimensional conformation of ADDLs despite similar linear
sequence recognition and affinities. Since ADDLs are known to
associate with a subset of neurons and disrupt normal neuronal
function, the antibodies of this invention find use in the
prevention of ADDL binding to neurons and the assembly of ADDLs
and, in turn, can be used for the treatment of ADDL-related
diseases including Alzheimer's disease.
[0066] Accordingly, one embodiment of the present invention is an
isolated antibody that differentially recognizes one or more
multi-dimensional conformations of ADDLs. An "isolated" antibody of
the present invention refers to an antibody which is substantially
free of other antibodies. However, the molecule may include some
additional agents or moieties which do not deleteriously affect the
basic characteristics of the antibody (for example, binding
specificity, neutralizing activity, etc.).
[0067] An antibody which is capable of specifically binding one or
more multi-dimensional conformations of ADDLs, binds particular
ADDLs derived from the oligomerization of A.beta.1-42, but does not
cross-react with other A.beta. peptides, namely A.beta.1-12,
A.beta.1-28, A.beta.1-40, and A.beta.12-28 as determined by western
blot analyses as disclosed herein, and preferentially binds ADDLs
in solution. Specific binding between two entities generally refers
to an affinity of at least 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9,
or 10.sup.10M.sup.-1. Affinities greater than 10.sup.8 M.sup.-1 are
desired to achieve specific binding.
[0068] In particular embodiments, an antibody that is capable of
specifically binding a multi-dimensional conformation of one or
more ADDLs is also raised against, i.e., an animal is immunized
with, multi-dimensional conformations of ADDLs. In other
embodiments, an antibody that is capable of specifically binding a
multi-dimensional conformation of one or more ADDLs is raised
against a low n-mer-forming peptide such as
A.beta.1-42[Nle35-Dpro37].
[0069] The term "epitope" refers to a site on an antigen to which B
and/or T cells respond or a site on a molecule against which an
antibody will be produced and/or to which an antibody will bind.
For example, an epitope can be recognized by an antibody defining
the epitope.
[0070] A linear epitope is an epitope wherein an amino acid primary
sequence comprises the epitope recognized. A linear epitope
typically includes at least 3, and more usually, at least 5, for
example, about 6 to about 10 amino acids in a unique sequence.
[0071] A conformational epitope, in contrast to a linear epitope,
is an epitope wherein the primary sequence of the amino acids
comprising the epitope is not the sole defining component of the
epitope recognized (for example, an epitope wherein the primary
sequence of amino acids is not necessarily recognized by the
antibody defining the epitope). Typically a conformational epitope
encompasses an increased number of amino acids relative to a linear
epitope. With regard to recognition of conformational epitopes, the
antibody recognizes a three-dimensional structure of the peptide or
protein. For example, when a protein molecule folds to form a
three-dimensional structure, certain amino acids and/or the
polypeptide backbone forming the conformational epitope become
juxtaposed enabling the antibody to recognize the epitope. Methods
of determining conformation of epitopes include but are not limited
to, for example, x-ray crystallography, two-dimensional nuclear
magnetic resonance spectroscopy and site-directed spin labeling and
electron paramagnetic resonance spectroscopy. See, for example,
Epitope Mapping Protocols in Methods in Molecular Biology (1996)
Vol. 66, Morris (Ed.).
[0072] Amyloid .beta.-derived diffusible ligands or ADDLs refer to
soluble oligomers of A.beta.1-42 which are desirably composed of
aggregates of less than eight or nine A.beta.1-42 peptides and are
found associated with Alzheimer's disease. This is in contrast to
high molecular weight aggregation intermediates, which form strings
of micelles leading to fibril formation.
[0073] As exemplified herein, the antibodies of the present
invention bind or recognize at least one multi-dimensional
conformation of an ADDL. In particular embodiments, the antibodies
bind at least two, at least three, or at least four
multi-dimensional conformations of an ADDL. Multi-dimensional
conformations of ADDLs are intended to encompass dimers, trimers,
tetramers pentamers, hexamers, heptamers, octamers, nonamers,
decamers, etc. as defined by analysis via SDS-PAGE. Because trimer,
tetramer, etc. designations can vary with the assay method employed
(see, e.g., Bitan, et al., 2005, Amyloid, 12:88-95), the definition
of trimer, tetramer, and the like, as used herein, is according to
SDS-PAGE analysis. To illustrate the differential binding
capabilities of the antibodies herein, it has been found that
certain antibodies will recognize one multi-dimensional
conformation, for example, tetramers of ADDLs (U.S. Pat. No.
7,780,963, murine antibodies 2D6 and 4E2), while other antibodies
recognize several multi-dimensional conformations, for example,
trimers and tetramers of ADDLs (U.S. Pat. No. 7,780,963, murine
antibodies 2A10, 2B4, 5F10, and 20C2 and humanized antibody 20C2).
As such, the antibody of the present invention has
oligomer-specific characteristics. In particular embodiments, a
multi-dimensional conformation of an ADDL is associated with a
specific polypeptide structure which results in a conformational
epitope that is recognized by an antibody of the present invention.
In other embodiments, an antibody of the invention specifically
binds a multi-dimensional conformation ADDL having a size range of
approximately a trimer or tetramer, which have molecular weights in
excess of >50 kDa.
[0074] While antibodies of the present invention may have similar
linear epitopes, such linear epitopes are not wholly indicative of
the binding characteristics of these antibodies, i.e., ability to
block ADDL binding to neurons, prevent tau phosphorylation and
inhibit ADDL assembly, because, as is well-known to the skilled
artisan, the linear epitope may only correspond to a portion of the
antigen's epitope (see, for example, Breitling and Dubel, 1999,
Recombinant Antibodies, John Wiley & Sons, Inc., NY, pg. 115).
The antibodies of the present invention can be distinguished from
those of the art as being capable of differentially recognizing
multi-dimensional ADDLs and accordingly differentially blocking
ADDL binding to neurons, differentially preventing tau
phosphorylation and differentially inhibiting ADDL assembly.
[0075] An antibody, as used in accordance with the present
invention includes, but is not be limited to, polyclonal or
monoclonal antibodies, and chimeric, human (for example, isolated
from B cells), humanized, neutralizing, bispecific or single chain
antibodies thereof. In one embodiment, an antibody of the present
invention is monoclonal. For the production of antibodies, various
hosts including goats, rabbits, chickens, rats, mice, humans, and
others, can be immunized by injection with synthetic or natural
ADDLs. Methods for producing antibodies are well-known in the art.
See, for example, Kohler and Milstein, 1975, Nature, 256:495-497:
Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory, New York, 1988.
[0076] Depending on the host species, various adjuvants can be used
to increase the immunological response. Adjuvants used in
accordance with the present invention desirably augment the
intrinsic response to ADDLs without causing conformational changes
in the immunogen that affect the qualitative form of the response.
Particularly suitable adjuvants include 3 De-O-acylated
monophosphoryl lipid A (MPL.TM.; RIBI ImmunoChem Research Inc.,
Hamilton, Mont.; see GB 2220211) and oil-in-water emulsions, such
as squalene or peanut oil, optionally in combination with immune
stimulants, such as monophosphoryl lipid A (see, Stoute, et al.,
1997, N. Engl. J. Med., 336:86-91), muramyl peptides (for example,
N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'dipalmitoyl-sn-
-glycero-3-hydroxyphosphoryloxy)-ethylamine (E-PE),
N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxy
propylamide (DTP-DPP)), or other bacterial cell wall components.
Specific examples of oil-in-water emulsions include MF59 (WO
90/14837), containing 5% Squalene, 0.5% TWEEN.TM. 80, and 0.5% SPAN
85 (optionally containing various amounts of MTP-PE) formulated
into submicron particles using a microfluidizer such as Model 110Y
microfluidizer (Microfluidics, Newton, Mass.); SAF containing 10%
Squalene, 0.4% TWEEN.TM. 80, 5% PLURONIC.RTM.-blocked polymer L121,
and thr-MDP, either microfluidized into a submicron emulsion or
vortexed to generate a larger particle size emulsion; and RIBI.TM.
adjuvant system (RAS) (Ribi ImmunoChem, Hamilton, Mont.) containing
2% squalene, 0.2% TWEEN.TM. 80, and one or more bacterial cell wall
components such as monophosphoryllipid A, trehalose dimycolate
(TDM), and cell wall skeleton (CWS).
[0077] Another class of adjuvants is saponin adjuvants, such as
STIMULON.TM. (QS-21, Aquila, Framingham, Mass.) or particles
generated therefrom such as ISCOMs (immunostimulating complexes)
and ISCOMATRIX.RTM. (CSL Ltd., Parkville, Australia). Other
suitable adjuvants include Complete Freund's Adjuvant (CFA),
Incomplete Freund's Adjuvant (IFA), mineral gels such as aluminum
hydroxide, and surface-active substances such as lysolecithin,
PLURONIC.RTM. polyols, polyanions, peptides, CpG (WO 98/40100),
keyhole limpet hemocyanin, dinitrophenol, and cytokines such as
interleukins (IL-1, IL-2, and IL-12), macrophage colony stimulating
factor (M-CSF), and tumor necrosis factor (TNF). Among adjuvants
used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium
parvum are particularly suitable.
[0078] An antibody to a multi-dimensional conformation ADDL is
generated by immunizing an animal with ADDLs. Generally, ADDLs can
be generated synthetically or by recombinant fragment expression
and purification. Synthetic ADDLs can be prepared as disclosed
herein, or in accordance with the methods disclosed in U.S. Pat.
Nos. 6,218,506 and 7,811,563, or in co-pending applications U.S.
2007/0218499, U.S. 2010/0143396, and U.S. 2010/0240868, all of
which are incorporated herein by reference in their entirety.
Further, ADDLs can be fused with another protein such as keyhole
limpet hemocyanin to generate an antibody against the chimeric
molecule. The ADDLs can be conformationally constrained to form an
epitope useful as described herein and furthermore can be
associated with a surface for example, physically attached or
chemically bonded to a surface in such a manner so as to allow for
the production of a conformation which is recognized by the
antibodies of the present invention.
[0079] Monoclonal antibodies to multi-dimensional conformations of
ADDLs can be prepared using any technique which provides for the
production of antibody molecules by continuous cell lines in
culture. These include, but are not limited to, the hybridoma
technique, the human B-cell hybridoma technique, and the
EBV-hybridoma technique (Kohler, et al., 1975, Nature 256:495-497;
Kozbor, et al., 1985, J. Immunol. Methods 81:31-42; Cote, et al.,
1983, Proc. Natl. Acad. Sci. 80:2026-2030; Cole, et al., 1984, Mol.
Cell Biol. 62:109-120).
[0080] In particular embodiments, the antibodies of the present
invention are humanized. Humanized or chimeric antibodies can be
produced by splicing of mouse antibody genes to human antibody
genes to obtain a molecule with appropriate antigen specificity and
biological activity (see, Morrison, et al., 1984, Proc. Natl. Acad.
Sci. 81, 6851-6855; Neuberger, et al., 1984, Nature 312:604-608;
Takeda, et al., 1985, Nature 314:452-454; Queen, et al., 1989,
Proc. Natl. Acad. Sci. USA 86:10029-10033; WO 90/07861). For
example, a mouse antibody is expressed as the Fv or Fab fragment in
a phage selection vector. The gene for the light chain (and in a
parallel experiment, the gene for the heavy chain) is exchanged for
a library of human antibody genes. Phage antibodies, which still
bind the antigen, are then identified. This method, commonly known
as chain shuffling, provided humanized antibodies that should bind
the same epitope as the mouse antibody from which it descends
(Jespers, et al., 1994, Biotechnology NY 12:899-903). As an
alternative, chain shuffling can be performed at the protein level
(see, Figini, et al., 1994, J. Mol. Biol. 239:68-78).
[0081] Human antibodies can also be obtained using phage-display
methods. See, for example, WO 91/17271 and WO 92/01047. In these
methods, libraries of phage are produced in which members display
different antibodies on their outer surfaces. Antibodies are
usually displayed as Fv or Fab fragments. Phage displaying
antibodies with a desired specificity are selected by affinity
enrichment to ADDLs. Human antibodies against ADDLs can also be
produced from non-human transgenic mammals having transgenes
encoding at least a segment of the human immunoglobulin locus and
an inactivated endogenous immunoglobulin locus. See, for example,
WO 93/12227 and WO 91/10741. Human antibodies can be selected by
competitive binding experiments, or otherwise, to have the same
epitope specificity as a particular mouse antibody. Such antibodies
generally retain the useful functional properties of the mouse
antibodies. Human polyclonal antibodies can also be provided in the
form of serum from humans immunized with an immunogenic agent.
Optionally, such polyclonal antibodies can be concentrated by
affinity purification using ADDLs as an affinity reagent.
[0082] As exemplified herein, humanized antibodies can also be
produced by veneering or resurfacing of murine antibodies.
Veneering involves replacing only the surface fixed region amino
acids in the mouse heavy and light variable regions with those of a
homologous human antibody sequence. Replacing mouse surface amino
acids with human residues in the same position from a homologous
human sequence has been shown to reduce the immunogenicity of the
mouse antibody while preserving its ligand binding. The replacement
of exterior residues generally has little, or no, effect on the
interior domains, or on the inter-domain contacts. See, for
example, U.S. Pat. No. 6,797,492.
[0083] Human or humanized antibodies can be designed to have IgG,
IgD, IgA, IgM or IgE constant regions, and any isotype, including
IgG1, IgG2, IgG3 and IgG4. In particular embodiments, an antibody
of the invention is IgG or IgM, or a combination thereof. In one
specific embodiment the antibodies of the present invention are
IgG2. Those of skill in the art would understand that other
isoforms can be utilized herein. Exemplary sequences for these
isoforms are given in SEQ ID NOS: 43-45. Other embodiments of the
present invention embrace a constant region formed by selective
incorporation of human IgG4 sequences into a standard human IgG2
constant region. An exemplary mutant IgG2 Fc is IgG2m4, set forth
herein as SEQ ID NO: 46. Antibodies can be expressed as tetramers
containing two light and two heavy chains, as separate heavy chains
and light chains or as single chain antibodies in which heavy and
light chain variable domains are linked through a spacer.
Techniques for the production of single chain antibodies are
well-known in the art.
[0084] Exemplary humanized antibodies produced by CDR grafting and
veneering are disclosed in U.S. Pat. Nos. 7,780,963, 7,731,962, and
7,811,563.
[0085] Diabodies are also contemplated. A diabody refers to an
engineered antibody construct prepared by isolating the binding
domains (both heavy and light chain) of a binding antibody, and
supplying a linking moiety which joins or operably links the heavy
and light chains on the same polypeptide chain thereby preserving
the binding function (see, Holliger, et al., 1993, Proc. Natl.
Acad. Sci. USA 90:6444; Poljak, 1994, Structure 2:1121-1123). This
forms, in essence, a radically abbreviated antibody, having only
the variable domain necessary for binding the antigen. By using a
linker that is too short to allow pairing between the two domains
on the same chain, the domains are forced to pair with the
complementary domains of another chain and create two
antigen-binding sites. These dimeric antibody fragments, or
diabodies, are bivalent and bispecific. The skilled artisan will
appreciate that any method to generate diabodies can be used.
Suitable methods are described by Holliger, et al., 1993, supra;
Poljak, 1994, supra; Zhu, et al., 1996, Biotechnology 14:192-196,
and U.S. Pat. No. 6,492,123, which are incorporated herein by
reference in their entirety.
[0086] Fragments of an isolated antibody of the invention are also
expressly encompassed by the present invention. Fragments are
intended to include Fab fragments, F(ab').sub.2 fragments, F(ab')
fragments, bispecific scFv fragments, Fv fragments and fragments
produced by a Fab expression library, as well as peptide aptamers.
For example, F(ab').sub.2 fragments are produced by pepsin
digestion of the antibody molecule of the invention, whereas Fab
fragments are generated by reducing the disulfide bridges of the
F(ab').sub.2 fragments. Alternatively, Fab expression libraries can
be constructed to allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity (see, Huse, et al.,
1989, Science, 254:1275-1281). In particular embodiments, antibody
fragments of the present invention are fragments of neutralizing
antibodies which retain the variable region binding site thereof,
i.e. antigen binding fragment. Exemplary are F(ab').sub.2
fragments, F(ab') fragments, and Fab fragments. See, generally,
Immunology: Basic Processes, 1985, 2.sup.nd edition, J. Bellanti
(Ed.) pp. 95-97.
[0087] Peptide aptamers which differentially recognize
multi-dimensional conformations of ADDLs can be rationally designed
or screened for in a library of aptamers (for example, provided by
Aptanomics SA, Lyon, France). In general, peptide aptamers are
synthetic recognition molecules whose design is based on the
structure of antibodies. Peptide aptamers consist of a variable
peptide loop attached at both ends to a protein scaffold. This
double structural constraint greatly increases the binding affinity
of the peptide aptamer to levels comparable to that of an antibody
(nanomolar range).
[0088] Exemplary nucleic acid sequences encoding heavy and light
chain variable regions for use in producing antibody and antibody
fragments of the present invention are disclosed herein in SEQ ID
NOS: 14 and 16. As will be appreciated by the skilled artisan, the
heavy chain variable regions disclosed herein, such as that shown
in SEQ ID NO: 16, can be used in combination with any one of the
light chain variable regions disclosed herein to generate
antibodies with modified affinities, dissociation, epitopes, and
the like.
[0089] Antibodies or antibody fragments of the present invention
can have additional moieties attached thereto. For example, a
microsphere or microparticle can be attached to the antibody or
antibody fragment, as described in U.S. Pat. No. 4,493,825, the
disclosure of which is incorporated herein by reference in its
entirety.
[0090] Moreover, particular embodiment embrace antibody or antibody
fragments which are mutated and selected for increased antigen
affinity, neutralizing activity (i.e., the ability to block binding
of ADDLs to neuronal cells or the ability to block ADDL assembly),
or a modified dissociation constant. Mutator strains of E. coli
(Low, et al., 1996, J. Mol. Biol., 260:359-368), chain shuffling
(Figini, et al., 1994, supra), and PCR mutagenesis are established
methods for mutating nucleic acid molecules encoding antibodies. By
way of illustration, increased affinity can be selected for by
contacting a large number of phage antibodies with a low amount of
biotinylated antigen so that the antibodies compete for binding. In
this case, the number of antigen molecules should exceed the number
of phage antibodies, but the concentration of antigen should be
somewhat below the dissociation constant. Thus, predominantly
mutated phage antibodies with increased affinity bind to the
biotinylated antigen, while the larger part of the weaker affinity
phage antibodies remains unbound. Streptavidin can then assist in
the enrichment of the higher affinity, mutated phage antibodies
from the mixture (Schier, et al., 1996, J. Mol. Biol. 255:28-43).
Exemplary affinity-maturated light chain CDR3 amino acid sequences
are disclosed herein (see Table 4), with particular embodiments
embracing a light chain CDR3 amino acid sequence of SEQ ID NO: 3
and specific embodiments of SEQ ID NOS: 7-13. The present invention
also embraces alternative variations for light chain CDR1 (SEQ ID
NO: 53) and CDR2 (SEQ ID NO: 54).
[0091] For some therapeutic applications it may be desirable to
reduce the dissociation of the antibody from the antigen. To
achieve this, phage antibodies are bound to biotinylated antigen
and an excess of unbiotinylated antigen is added. After a period of
time, predominantly the phage antibodies with the lower
dissociation constant can be harvested with streptavidin (Hawkins,
et al., 1992, J. Mol. Biol. 226:889-96).
[0092] Various immunoassays including those disclosed herein can be
used for screening to identify antibodies, or fragments thereof,
having the desired specificity for multi-dimensional conformations
of ADDLs. Numerous protocols for competitive binding (for example,
ELISA), latex agglutination assays, immunoradiometric assays,
kinetics (for example, Biacore.TM. analysis) using either
polyclonal or monoclonal antibodies, or fragments thereof, are
well-known in the art. Such immunoassays typically involve the
measurement of complex formation between a specific antibody and
its cognate antigen. A two-site, monoclonal-based immunoassay
utilizing monoclonal antibodies reactive to two non-interfering
epitopes is suitable, but a competitive binding assay can also be
employed. Such assays can also be used in the detection of
multi-dimensional conformations of ADDLs in a sample.
[0093] An antibody or antibody fragment can also be subjected to
other biological activity assays, e.g., displacement of ADDL
binding to neurons or cultured hippocampal cells or blockade of
ADDL assembly, in order to evaluate neutralizing or pharmacological
activity and potential efficacy as a prophylactic or therapeutic
agent. Such assays are described herein and are well-known in the
art.
[0094] Antibodies and fragments of antibodies can be produced and
maintained as hybridomas or, alternatively, recombinantly produced
in any well-established expression system including, but not
limited to, E. coli, yeast (e.g., Saccharomyces spp. and Pichia
spp.), baculovirus, mammalian cells (e.g., myeloma, CHO, COS),
plants, or transgenic animals (Breitling and Dubel, 1999,
Recombinant Antibodies, John Wiley & Sons, Inc., NY, pp.
119-132). Antibodies and fragments of antibodies can be isolated
using any appropriate methods including, but not limited to,
affinity chromatography, immunoglobulins-binding molecules (for
example, proteins A, L, G or H), tags operatively linked to the
antibody or antibody fragment (for example, His-tag, FLAG.RTM.-tag,
Strep tag, c-myc tag) and the like. See, Breitling and Dubel, 1999
supra.
[0095] Antibodies and antibody fragments of the present invention
have a variety of uses including, diagnosis of diseases associated
with accumulation of ADDLs, blocking or inhibiting binding of ADDLs
to neuronal cells, blocking ADDL assembly, prophylactically or
therapeutically treating a disease associated with ADDLs,
identifying therapeutic agents that prevent binding of ADDLs to
neurons, and preventing the phosphorylation of tau protein at
Ser202/Thr205.
[0096] Antibody and antibody fragments of the present invention are
useful in a method for blocking or inhibiting binding of ADDLs to
neuronal cells. This method of the invention is carried out by
contacting a neuron, in vitro or in vivo, with an antibody or
antibody fragment of the present invention so that binding of ADDLs
to the neuron is blocked. In particular embodiments, an antibody or
antibody fragment of the present invention achieves at least a 15%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 97% decrease in the
binding of ADDLs as compared to binding of ADDLs in the absence of
the antibody or antibody fragment. The degree to which an antibody
can block the binding of ADDLs to a neuron can be determined in
accordance with the methods disclosed herein, i.e.,
immunocytochemistry, or cell-based alkaline phosphatase assay, or
any other suitable assay. Antibodies particularly useful for
decreasing binding of ADDLs to neuronal cells include the exemplary
anti-ADDL antibodies shown in U.S. Pat. Nos. 7,731,962, 7,780,963,
and 7,811,563, as well as derivatives and fragments thereof.
[0097] Antibody and antibody fragments of the present invention are
further useful in a method for blocking or inhibiting assembly of
ADDLs. This method involves contacting a sample containing amyloid
.beta.1-42 peptides with an antibody or antibody fragment of the
present invention so that ADDL assembly is inhibited. The degree to
which an antibody can block the assembly of ADDLs can be determined
in accordance with the methods disclosed herein, i.e., FRET or
fluorescence polarization or any other suitable assay. Antibodies
particularly useful for blocking the assembly of ADDLs include
anti-ADDL antibodies having a CDR3 amino acid sequence set forth in
SEQ ID NO: 10, as well as derivatives and fragments thereof.
[0098] Antibodies disclosed herein are also useful in methods for
preventing the phosphorylation of tau protein at Ser202/Thr205.
This method involves contacting a sample containing tau protein
with an antibody or antibody fragment of the present invention so
that binding of ADDLs to neurons is blocked thereby preventing
phosphorylation of tau protein. The degree to which an antibody can
prevent the phosphorylation of tau protein at Ser202/Thr205 can be
determined in accordance with the methods disclosed herein or any
other suitable assay.
[0099] Blocking or decreasing binding of ADDLs to neurons,
inhibiting assembly of ADDLs, and preventing the phosphorylation of
tau protein at Ser202/Thr205 all find application in methods of
prophylactically or therapeutically treating a disease associated
with the accumulation of ADDLs. Accordingly, the present invention
also embraces the use of an antibody or antibody fragment herein to
prevent or treat a disease associated with the accumulation of
ADDLs (for example, Alzheimer's disease or similar memory-related
disorders). Evidence in the art indicates that elevated levels of
A.beta., but not necessarily aggregated plaque, cause Alzheimer's
disease-associated dementia and subsequent tau abnormalities.
A.beta.-derived diffusible ligands are directly implicated in
neurotoxicity associated with Alzheimer's disease. The art
indicates that ADDLs are elevated in transgenic mice and
Alzheimer's disease patients and modulate functional activity
associated with mnemonic processes in animal models. Thus, removing
this form of A.beta. could provide relief from the neurotoxicity
associated with Alzheimer's disease. As such, treatment with an
antibody of the present invention that reduces central nervous
system ADDL load could prove efficacious for the treatment of
Alzheimer's disease. Patients amenable to treatment include
individuals at risk of disease but not exhibiting symptoms, as well
as patients presently exhibiting symptoms. In the case of
Alzheimer's disease, virtually anyone is at risk of suffering from
Alzheimer's disease if he or she lives long enough. Therefore, the
antibody or antibody fragments of the present invention can be
administered prophylactically to the general population without the
need for any assessment of the risk of the subject patient. The
present methods are especially useful for individuals who have a
known genetic risk of Alzheimer's disease. Such individuals include
those having relatives who have been diagnosed with the disease,
and those whose risk is determined by analysis of genetic or
biochemical markers. Genetic markers of risk for Alzheimer's
disease include mutations in the APP gene, particularly mutations
at position 717 and positions 670 and 671 referred to as the Hardy
and Swedish mutations respectively. Other markers of risk are
mutations in the presenilin genes, PS1 and PS2, and ApoE4, family
history of Alzheimer's disease, hypercholesterolemia or
atherosclerosis. Individuals presently suffering from Alzheimer's
disease can be recognized from characteristic dementia, as well as
the presence of risk factors described above. In addition, a number
of diagnostic tests are available for identifying individuals who
have Alzheimer's disease. These include measurement of CSF tau and
A.beta.1-42 levels. Individuals suffering from Alzheimer's disease
can also be diagnosed by ADRDA criteria or the method disclosed
herein.
[0100] In asymptomatic patients, treatment can begin at any age
(for example, 10, 20, 30 years of age). Usually, however, it is not
necessary to begin treatment until a patient reaches 40, 50, 60 or
70 years of age. Treatment typically entails multiple dosages over
a period of time. Treatment can be monitored by assaying for the
presence of ADDLs over time.
[0101] In therapeutic applications, a pharmaceutical composition or
medicament containing an antibody or antibody fragment of the
invention is administered to a patient suspected of, or already
suffering from such a disease associated with the accumulation of
ADDLs in an amount sufficient to cure, or at least partially
arrest, the symptoms of the disease (biochemical, histologic and/or
behavioral), including its complications and intermediate
pathological phenotypes in development of the disease. In
prophylactic applications, a pharmaceutical composition or
medicament containing an antibody or antibody fragment of the
invention is administered to a patient susceptible to, or otherwise
at risk of, a disease associated with the accumulation of ADDLs in
an amount sufficient to achieve passive immunity in the patient
thereby eliminating or reducing the risk, lessening the severity,
or delaying the onset of the disease, including biochemical,
histologic and/or behavioral symptoms of the disease, its
complications and intermediate pathological phenotypes present
during development of the disease. In some methods, administration
of agent reduces or eliminates myocognitive impairment in patients
that have not yet developed characteristic Alzheimer's pathology.
In particular embodiments, an effective amount of an antibody or
antibody fragment of the invention is an amount which achieves at
least a 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 97%
decrease in the binding of ADDLs to neurons in the patient as
compared to binding of ADDLs in the absence of treatment. As such,
impairment of long-term potentiation/memory formation is
decreased.
[0102] Effective doses of the compositions of the present
invention, for the treatment of the above described conditions vary
depending upon many different factors, including means of
administration, physiological state of the patient, whether the
patient is human or an animal, other medications administered, and
whether treatment is prophylactic or therapeutic. Usually, the
patient is a human but nonhuman mammals such as dogs or transgenic
mammals can also be treated.
[0103] Treatment dosages are generally titrated to optimize safety
and efficacy. For passive immunization with an antibody or antibody
fragment, dosage ranges from about 0.0001 to 100 mg/kg, and more
usually 0.01 to 5 mg/kg, of the host body weight are suitable. For
example, dosages can be 1 mg/kg body weight or 10 mg/kg body weight
or within the range of 1-10 mg/kg. In some methods, two or more
antibodies of the invention with different binding specificities
are administered simultaneously, in which case the dosage of each
antibody administered falls within the ranges indicated. Antibodies
are usually administered on multiple occasions, wherein intervals
between single dosages can be weekly, monthly or yearly. An
exemplary treatment regime entails subcutaneous dosing, once
biweekly or monthly. Intervals can also be irregular as indicated
by measuring blood levels of antibody to ADDLs in the patient. In
some methods, dosage is adjusted to achieve a plasma antibody
concentration of 1-1000 .mu.g/mL and in some methods 25-300
.mu.g/mL. Alternatively, the antibody or antibody fragment can be
administered as a sustained-release formulation, in which case less
frequent administration is required. Dosage and frequency vary
depending on the half-life of the antibody in the patient. In
general, human and humanized antibodies have longer half-lives than
chimeric antibodies and nonhuman antibodies. As indicated above,
dosage and frequency of administration can vary depending on
whether the treatment is prophylactic or therapeutic. In
prophylactic applications, a relatively low dosage is administered
at relatively infrequent intervals over a long period of time. Some
patients continue to receive treatment for the rest of their lives.
In therapeutic applications, a relatively high dosage at relatively
short intervals is sometimes required until progression of the
disease is reduced or terminated, and preferably until the patient
shows partial or complete amelioration of symptoms of disease.
Thereafter, the patient can be administered a prophylactic
regime.
[0104] Antibody and antibody fragments of the present invention can
be administered as a component of a pharmaceutical composition or
medicament. Pharmaceutical compositions or medicaments generally
contain the active therapeutic agent and a variety of other
pharmaceutically acceptable components. See, Remington: The Science
and Practice of Pharmacy, Alfonso R. Gennaro, editor, 20th ed.
Lippincott Williams & Wilkins: Philadelphia, Pa., 2000. The
preferred form depends on the intended mode of administration and
therapeutic application. Pharmaceutical compositions can contain,
depending on the formulation desired, pharmaceutically-acceptable,
non-toxic carriers or diluents, which are defined as vehicles
commonly used to formulate pharmaceutical compositions for animal
or human administration. Diluents are selected so as not to affect
the biological activity of the combination. Examples of such
diluents are distilled water, physiological phosphate-buffered
saline, Ringer's solutions, dextrose solution, and Hank's
solution.
[0105] Pharmaceutical compositions can also contain large, slowly
metabolized macromolecules such as proteins, polysaccharides such
as chitosan, polylactic acids, polyglycolic acids and copolymers
(such as latex-functionalized SEPHAROSE.TM., agarose, cellulose,
and the like), polymeric amino acids, amino acid copolymers, and
lipid aggregates (such as oil droplets or liposomes).
[0106] Administration of a pharmaceutical composition or medicament
of the invention can be carried out in a variety of routes
including, but not limited to, oral, topical, pulmonary, rectal,
subcutaneous, intradermal, intranasal, intracranial, intramuscular,
intraocular, or intrathecal or intra-articular injection, and the
like. The most typical route of administration is intravenous
followed by subcutaneous, although other routes can be equally
effective. Intramuscular injection can also be performed in the arm
or leg muscles. In some methods, agents are injected directly into
a particular tissue where deposits have accumulated, for example,
intracranial or intrathecal injection. In some embodiments, an
antibody or antibody fragment is injected directly into the cranium
or CSF. In other embodiments, antibody or antibody fragment is
administered as a sustained-release composition or device, such as
a MEDIPAD.TM. device.
[0107] For parenteral administration, antibody or antibody
fragments of the invention can be administered as injectable
dosages of a solution or suspension of the substance in a
physiologically acceptable diluent with a pharmaceutical carrier
that can be a sterile liquid such as water, oils, saline, glycerol,
or ethanol. Additionally, auxiliary substances, such as wetting or
emulsifying agents, surfactants, pH buffering substances and the
like can be present in compositions. Other components of
pharmaceutical compositions are those of petroleum, animal,
vegetable, or synthetic origin, for example, peanut oil, soybean
oil, and mineral oil. In general, glycols such as propylene glycol
or polyethylene glycol are suitable liquid carriers, particularly
for injectable solutions. Antibodies can be administered in the
form of a depot injection or implant preparation which can be
formulated in such a manner as to permit a sustained-release of the
active ingredient.
[0108] An exemplary composition contains an isolated antibody, or
antibody fragment thereof, of the present invention formulated as a
sterile, clear liquid at a concentration of at least 10 mg/ml in
isotonic buffered saline (10 mM histidine, 150 mM sodium chloride,
0.01% (w/v) POLYSORBATE 80, pH 6.0). An exemplary antibody
formulation is filled as a single dose, 0.6 ml glass vials filled
with 3.3 ml of solution per vial. Each vial is stopped with a
TEFLON-coated stopper and sealed with an aluminum cap.
[0109] Typically, compositions are prepared as injectables, either
as liquid solutions or suspensions; solid forms suitable for
solution in, or suspension in, liquid vehicles prior to injection
can also be prepared. The preparation also can be emulsified or
encapsulated in liposomes or micro particles such as polylactide,
polyglycolide, or copolymer for enhanced delivery.
[0110] For suppositories, binders and carriers include, for
example, polyalkylene glycols or triglycerides; such suppositories
can be formed from mixtures containing the active ingredient in the
range of 0.5% to 10%, or more desirably 1%-2%.
[0111] Oral formulations include excipients, such as pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, and magnesium carbonate. These compositions
take the form of solutions, suspensions, tablets, pills, capsules,
sustained-release formulations or powders and contain 10%-95% of
active ingredient, or more suitably 25%-70%.
[0112] Topical application can result in transdermal or intradermal
delivery. Topical administration can be facilitated by
co-administration of the agent with cholera toxin or detoxified
derivatives or subunits thereof or other similar bacterial toxins
(see Glenn, et al. (1998) Nature 391:851). Co-administration can be
achieved by using the components as a mixture or as linked
molecules obtained by chemical crosslinking or expression as a
fusion protein.
[0113] Alternatively, transdermal delivery can be achieved using a
skin path or using transferosomes (Paul, et al., 1995, Eur. J.
Immunol. 25:3521-3524; Cevc, et al., 1998, Biochem. Biophys. Acta
1368:201-215).
[0114] An antibody or antibody fragment of the invention can
optionally be administered in combination with other agents that
are at least partly effective in treatment of amyloidogenic
disease. For example, the present antibody can be administered with
existing palliative treatments for Alzheimer's disease, such as
acetylcholinesterase inhibitors such as ARICEPT.TM., EXELON.TM.,
and REMINYL.TM. and, the NMDA antagonist, NAMENDA.TM.. In addition
to these approved treatments, the present antibody can be used to
provide synergistic/additive benefit for any of several approaches
currently in development for the treatment of Alzheimer's disease,
which include without limitation, inhibitors of A.beta. production
and aggregation.
[0115] Antibody and antibody fragments of the present invention
also find application in the identification of therapeutic agents
that prevent the binding of ADDLs to neurons (e.g., a hippocampal
cell) thereby preventing downstream events attributed to ADDLs.
Such an assay is carried out by contacting a neuron with ADDLs in
the presence of an agent and using an antibody of antibody fragment
of the invention to determine binding of the ADDLs to the neuron in
the presence of the agent. As will be appreciated by the skilled
artisan, an agent that blocks binding of ADDLs to a neuron will
decrease the amount of ADDLs bound to the neuron as compared to a
neuron which has not been contacted with the agent; an amount which
is detectable in an immunoassay employing an antibody or antibody
fragment of the present invention. Suitable immunoassays for
detecting neuronal-bound ADDLs are disclosed herein.
[0116] Agents which can be screened using the method provided
herein encompass numerous chemical classes, although typically they
are organic molecules, preferably small organic compounds having a
molecular weight of more than 100 and less than about 2,500
daltons. Agents encompass functional groups necessary for
structural interaction with proteins, particularly hydrogen
bonding, and typically include at least an amine, carbonyl,
hydroxyl or carboxyl group, preferably at least two of the
functional chemical groups. The agents often contain cyclical
carbon or heterocyclic structures and/or aromatic or polyaromatic
structures substituted with one or more of the above functional
groups. Agents can also be found among biomolecules including
peptides, antibodies, saccharides, fatty acids, steroids, purines,
pyrimidines, derivatives, structural analogs or combinations
thereof. Agents are obtained from a wide variety of sources
including libraries of natural or synthetic compounds.
[0117] A variety of other reagents such as salts and neutral
proteins can be included in the screening assays. Also, reagents
that otherwise improve the efficiency of the assay, such as
protease inhibitors, nuclease inhibitors, anti-microbial agents,
and the like can be used. The mixture of components can be added in
any order that provides for the requisite binding.
[0118] Agents identified by the screening assay of the present
invention will be beneficial for the treatment of amyloidogenic
diseases and/or tauopathies. In addition, it is contemplated that
the experimental systems used to exemplify these concepts represent
research tools for the evaluation, identification and screening of
novel drug targets associated with amyloid beta induction of tau
phosphorylation.
[0119] The present invention also provides methods for detecting
ADDLs and diagnosing a disease associated with accumulation of
ADDLs using an antibody or antibody fragment herein. A disease
associated with accumulation of ADDLs is intended to include any
disease wherein the accumulation of ADDLs results in physiological
impairment of long-term potentiation/memory formation. Diseases of
this type include, but are not limited to, Alzheimer's disease and
similar memory-related disorders.
[0120] In accordance with these methods, a sample from a patient is
contacted with an antibody or antibody fragment of the invention
and binding of the antibody or antibody fragment to the sample is
indicative of the presence of ADDLs in the sample. As used in the
context of the present invention, a sample is intended to mean any
bodily fluid or tissue which is amenable to analysis using
immunoassays. Suitable samples which can be analyzed in accordance
with the methods of the invention include, but are not limited to,
biopsy samples and fluid samples of the brain from a patient (for
example, a mammal such as a human). For in vitro purposes (for
example, in assays monitoring oligomer formation), a sample can be
a neuronal cell line or tissue sample. For diagnostic purposes, it
is contemplated that the sample can be from an individual suspected
of having a disease associated with accumulation of ADDLs or from
an individual at risk of having a disease associated with
accumulation of ADDLs, for example, an individual with a family
history which predisposes the individual to a disease associated
with accumulation of ADDLs.
[0121] Detection of binding of the antibody or antibody fragment to
ADDLs in the sample can be carried out using any standard
immunoassay (for example, as disclosed herein), or alternatively
when the antibody fragment is, for example, a peptide aptamer,
binding can be directly detected by, for example, a detectable
marker protein (for example, .beta.-galactosidase, GFP or
luciferase) fused to the aptamer. Subsequently, the presence or
absence of the ADDL-antibody complex is correlated with the
presence or absence, respectively, of ADDLs in the sample and
therefore the presence or absence, respectively, of a disease
associated with accumulation of ADDLs. It is contemplated that one
or more antibodies or antibody fragments of the present invention
can be used in conjunction with current non-invasive immuno-based
imaging techniques to greatly enhance detection and early diagnosis
of a disease associated with accumulation of ADDLs.
[0122] To facilitate diagnosis, the present invention also pertains
to a kit containing an antibody or antibody fragment herein. The
kit includes a container holding one or more antibodies or antibody
fragments which recognize multi-dimensional conformation of ADDLs
and instructions for using the antibody for the purpose of binding
to ADDLs to form an antibody-antigen complex and detecting the
formation of the antibody-antigen complex such that the presence or
absence of the antibody-antigen complex correlates with presence or
absence of ADDLs in the sample. Examples of containers include
multiwell plates which allow simultaneous detection of ADDLs in
multiple samples.
[0123] All references cited herein are incorporated herein by
reference in their entirety.
[0124] The invention is described in greater detail by the
following non-limiting examples.
EXAMPLES
[0125] The following abbreviations are used herein: Ab: antibody;
A.beta.: amyloid beta protein; AD: Alzheimer's disease; ADDL:
amyloid-.beta.(A.beta.)-derived diffusible ligand; Ag: antigen;
APP: amyloid precursor protein; bADDLs: biotinylated ADDLs; CSF:
cerebrospinal fluid; DMSO: dimethyl sulfoxide; hAAP: human amyloid
precursor protein; HAT medium: hypoxanthine-aminopterin-thymidine
medium; HFIP: hexafluoro-2-propanol; IV: intravenous; LB agar:
lysogeny broth agar; SC: subcutaneous; PBS: phosphate buffered
saline; TEA: Triethylamine.
Example 1
General Materials and Methods
A. Generation of ADDL Monoclonal Antibodies
[0126] Soluble A.beta. oligomers, a species of which is referred to
herein as "synthetic" ADDLs, were mixed 1:1 with complete Freund's
adjuvant (first and second vaccination) or incomplete Freund's
adjuvant (all subsequent vaccinations) and were given by
subcutaneous (first two vaccinations) or intraperitoneal injection
into three mice in a total volume of 1 mL/mouse. Each injection
consisted of purified ADDLs equivalent to 194.+-.25 .mu.g total
protein. Mice were injected approximately every three weeks. After
six injections, one mouse died and its spleen was frozen. The
spleen from the mouse with the highest titer serum was then fused
with SP2/0 myeloma cells in the presence of polyethylene glycol and
plated out into six 96-well plates. The cells were cultured at
37.degree. C. with 5% CO.sub.2 for 10 days in 200 .mu.L of
hypoxanthine-aminopterin-thymidine (HAT) selection medium, which is
composed of an enriched synthetic medium, such as Iscove's Modified
Dulbecco's Medium (IMDM), (Sigma-Aldrich, St. Louis, Mo.),
supplemented with 10% fetal bovine serum (FBS), 1 .mu.g/mL
HYBRI-MAX.RTM. (azaserine-hypoxanthine; Sigma-Aldrich, MO), and 30%
conditioned media collected from SP2/0 cell culture. The cultures
were fed once with IMDM (Sigma-Aldrich, St. Louis, Mo.)
supplemented with 10% FBS on day 10, and the culture supernatants
were removed on day 14 to screen for positive wells in ELISA. The
positive cultures were further cloned by limiting dilutions with
probability of 0.3 cells per well. The positive clones were
confirmed in ELISA and further expanded. Monoclonal antibodies were
then produced and purified for use (QED Bioscience, San Diego,
Calif.).
B. Preparation of ADDLs and bADDLs
[0127] ADDLs were prepared using previously described methods
(Hepler, et al., 2006, Biochemistry, 45: 15157-15167; Shughrue, et
al., 2010, Neurobiol. Aging, 31: 189-202). Briefly, synthetic
A.beta.1-42 peptide (American Peptide, Sunnyvale, Calif.) was
dissolved in hexafluoro-2-propanol (HFIP) at a concentration of 10
mg/ml, and incubated at room temperature (RT) for one hour. The
peptide solution was dispensed into 50 .mu.l aliquots in
polypropylene 1.5 ml microcentrifuge tubes. The HFIP was removed
using a SpeedVac.RTM. (Thermo-Fisher Scientific, Waltham, Mass.),
and the resulting peptide films were stored desiccated at
-70.degree. C. until needed. A 0.5 mg dried HFIP film was dissolved
in 22 .mu.l of anhydrous dimethyl sulfoxide (DMSO) with agitation
for 10 minutes on a vortex mixer. Subsequently, 1 ml of cold Ham's
F12 media without phenol red (United Biosource, San Francisco,
Calif.) was added rapidly to the DMSO/peptide mixture. The tube was
capped, inverted to insure complete mixing and incubated overnight
at 4.degree. C. The next morning the samples were centrifuged for
ten minutes at 12,000.times.g in a Beckman microcentrifuge (Beckman
Coulter, Brea, Calif.) operated at 2-8.degree. C. The supernatant
was collected and filtered through ym 50 (50,000 kDa molecular
cutoff) Centricon.RTM. centrifugal filter (Millipore, Billerica,
Mass.) to enrich the oligomeric species. Biotinylated ADDLs
(bADDLs) were prepared using the same methods, but starting with
N-terminal biotinylated A.beta.1-42 peptide (American Peptide,
Sunnyvale, Calif.).
C. Monomer and Fibril Preparations
[0128] To generate monomer preparations, RT A.beta.1-40 or
A.beta.1-42 peptide film was dissolved in 2 mL of 25 mM borate
buffer (pH 8.5) per mg of peptide, divided into aliquots, and
frozen at -70.degree. C. until used. The fibril preparations were
made by adding 2 mL of 10 mM hydrochloric acid per mg of
A.beta.1-42 peptide film. The solution was mixed on a vortex mixer
at the lowest possible speed for five to ten minutes and the
resulting preparation was stored at 37.degree. C. for 18 to 24
hours before use.
D. Primary Neurons
[0129] Primary neuronal cultures were prepared from rat hippocampal
and/or cortical tissues purchased from BrainBits (Springfield,
Ill.). After dissociation, cells were plated at a 35,000 cells/well
in 96-well plates pre-coated with laminin and poly-D-lysine
(Corning Life Sciences, Lowell, Mass.). Cells were maintained at
37.degree. C. with 5% CO.sub.2 in media (Neurobasal supplemented
with 2% B27, 1% L-glutamine, and 1% pen/strep; Invitrogen,
Carlsbad, Calif.) for two-three weeks and then used for binding
studies.
E. Cell-Based ADDL Binding Assay
[0130] To measure the effect of anti-ADDL antibodies on blocking
ADDL binding, anti-ADDL antibodies were mixed with 500 nM bADDLs,
with the final antibody concentrations ranging from 1.8 nM to 450
nM. As a control, the same concentration of heat-denatured antibody
(98.degree. C. for 30 minutes) was mixed with bADDLs. The
antibody-bADDL mixtures were incubated in siliconized
microcentrifuge tubes (Fischer Scientific, Pittsburgh, Pa.) at
37.degree. C. for one hour with constant end-to-end rotation at a
low speed. The mixtures were then applied to primary hippocampal
and/or cortical cultures and incubated at 37.degree. C. for one
hour. The incubation was terminated by removing the culture medium.
Cells were subjected to fixation and post-fixation treatments as
described above. Cells were then incubated with streptavidin
conjugated with alkaline phosphate (AP) at 4.degree. C. overnight,
washed five times with PBS and reacted with the Tropix.RTM.
CDP.RTM.-Star chemiluminescent substrate (Life Technologies.TM.,
Carlsbad, Calif.) at room temperature for 30 minutes. The bADDL
binding intensity was measured and recorded with an EnVision.RTM.
microplate reader (PerkinElmer, Waltham, Mass.).
F. ELISA
[0131] Biotinylated ADDLs (bADDLs) or monomer A.beta.1-40 or
A.beta.1-42 was added to a high-capacity streptavidin-coated plate
(Sigma-Aldrich, St. Louis, Mo.) with 100 .mu.L per well of coating
reagent in PBS at 1 .mu.M and incubated for two hours at room
temperature. The plates were washed in PBS with 0.05% Tween (six
times) and then PBS alone (three times) prior to blocking wells
with 5% non-fat dry milk in PBS for one hour at room temperature.
The wells were then washed and a serial dilution of antibody
samples added to the plates and allowed to bind for two hours at
room temperature. After incubation and washing, the antibody
binding was detected with a goat anti-human IgG-Fc secondary
antibody conjugated to horse radish peroxidase (HRP) (1:1,000; one
hour at room temperature). The HRP label was visualized with
tetramethyl benzidine (Virolabs, Chantilly, Va.) as a substrate and
read at 450 nm on a microplate reader.
Example 2
Selection of Anti-ADDL Antibodies
A. Panning Humanized Antibody Library
[0132] An affinity mature library of a humanized anti-ADDL
antibody, h3B3, (See, U.S. 2006/0228349 and U.S. 2008/0175835) was
constructed in which part of the light chain CDR3 amino acid
sequences were subject to random mutagenesis. To cover the entire
CDR3 region, two sub-libraries were built. One library was composed
of the parental heavy chain variable region and mutated amino acids
in the left half of the light chain CDR3 and the other in the right
half of the light chain CDR3. A similar strategy was used for heavy
chain CDRs random mutagenesis with three sub-libraries.
[0133] Humanized 3B3 (h3B3) was subject to affinity maturation
using methods known in the art. The h3B3 variable regions were
cloned in a Fab display vector (pFab3D). In this vector, the
variable regions for heavy and light chains were in-frame inserted
to match the CH1 domain of the constant region and the kappa
constant region, respectively. In Fab3D, myc epitope and six
consecutive histidine amino acids follow the CH1 sequence, which is
then linked to the phage pIII protein for display. All positions in
the heavy and light chain CDR3s were randomly mutagenized using
degenerate oligonucleotide sequences built in the PCR primers. To
accommodate the physical size, the sub-libraries were constructed
with each focusing on 5-6 amino acids. The vector DNA of human 3B3
(H3B3) was used as template DNA to amplify both heavy and light
chains with the mutated PCR primers (Table 1). After PCR
amplification, the synthesized DNA fragments were run on a 1.3%
agarose gel, the primers removed and the variable fragments
digested with restriction enzymes: BsiWI and XbaI cloning sites for
light chain variable cloning, XhoI and ApaI for heavy chain
variable cloning.
TABLE-US-00001 TABLE 1 3B3 Affinity Maturation Forward PCR Library
Primer Reverse PCR Primers Light Chain SEQ ID NO: 22 SEQ ID NO: 23
Libraries SEQ ID NO: 24 Heavy Chain SEQ ID NO: 25 SEQ ID NO: 26
Libraries SEQ ID NO: 27
[0134] To construct an affinity maturation library in pFab3D phage
display vector, pFab3D-3B3 DNA was digested with the same pair of
the restriction enzymes, purified and the PCR fragments for heavy
or light chain variables ligated with T4 ligase (Invitrogen)
overnight at 16.degree. C. The ligation products were then
transfected into E. coli TG1 electroporation-competent cells
(Stratagene, Agilent Technologies, Santa Clara, Calif.) and
aliquots of the bacterial culture plated on LB agar-carbenicillin
(50 .mu.g/mL) plates to titer library size. The remaining cultures
were either plated on a large plate with carbenicillin and
incubated at 30.degree. C. overnight for E. coli library stock or
infected with helper phage M13K07 (Invitrogen, Carlsbad, Calif.,
10.sup.11 pfu/mL) by incubating at room temperature and 37.degree.
C. for ten minutes. Then 2YT medium with carbenicillin (50
.mu.g/mL) was added and incubated at 37.degree. C. for one hour
with shaking. Kanamycin (70 .mu.g/ml) was then added and the
cultures grown overnight at 30.degree. C. with shaking. The phage
culture supernatant was tittered and concentrated by precipitation
with 20% (v/v) PEG (polyethleneglycol)/NaCl, resuspended in PBS,
sterilized with a 0.22 .mu.m filter, and aliquots made for phage
library panning.
[0135] Phage library panning was then conducted as summarized in
Table 2.
TABLE-US-00002 TABLE 2 Panning Rounds Round 1 Round 2 Round 3 Round
4 Antigen 180 nM 60 nM 20 nM 10 nM concentration
Input phages from the Fab display phage libraries (100 .mu.l, about
10.sup.11-12 pfu) were blocked with 900 .mu.L of blocking solution
(3% non-fat dry milk in PBS) to reduce nonspecific binding to the
phage surface. Streptavidin-coated beads were prepared by
collecting 200 .mu.L of the bead suspension in a magnetic separator
and removing supernatants. The beads were then suspended in 1 mL of
blocking solution and put on a rotary mixer for 30 minutes. To
remove non-specific streptavidin binding phage, the blocked phage
library was mixed with the blocked streptavidin-coated beads and
placed on a rotary mixer for thirty minutes. Phage suspensions from
the de-selection process were transferred to a new tube and 200
.mu.L of antigen, 10% bADDL was added and incubated for two hours
for antibody and antigen binding. After the incubation, the mixture
was added into the blocked Streptavidin-coated beads and incubated
on a rotary mixer for one hour to capture the Ab/Ag complex on
streptavidin beads. The beads with captured 10% bADDL/phage
complexes were washed five times with PBS/0.05% Tween 20 and then
twice with PBS alone. The bound phages were eluted from the bADDL
with 200 .mu.L of 100 mM TEA (Sigma Aldrich, St. Louis, Mo.) and
incubated for twenty minutes. The eluted phage were then
transferred to a 50 mL tube, neutralized with 100 .mu.L of 1M
Tris-HCl, pH7.5, and added to 10 mL of E. coli TG1 cells with an OD
600 nm between 0.6-0.8. After incubation at 37.degree. C. with
shaking for one hour, culture aliquots were plated on LB
agar-carbenicillin (50 .mu.g/mL) plates to titer the output phage
number, and the remaining bacteria centrifuged and suspended with
500 .mu.l 2xYT medium (Teknova, Hollister, Calif., plated on
bioassay YT agar plates (Teknova, Hollister, Calif.) containing 100
.mu.g/ml ampicillin and 1% glucose. The bioassay plates were grown
overnight at 30.degree. C.
[0136] After each round of panning, single colonies were randomly
picked to produce phage in 96-well plates. The procedures for phage
preparation in 96-well plate were similar to that described above
except no phage precipitation step was used. Culture plates
containing colonies growing in 120 .mu.L of 2.times.TY medium with
100 .mu.g/ml ampicillin and 0.1% glucose were incubated overnight
in a HiGro.RTM. shaker (Genomic Solutions, Ann Arbor, Mich.) at
30.degree. C. with shaking at 450 rpm. The phage supernatants
(about 100 .mu.L) were directly used for analysis in the ADDL
binding ELISA described above. One difference is that the binding
of phage to ADDLs was detected with an anti-M13-antibody conjugated
to HRP (Amersham Bioscience, GE Healthcare, Waukesha, Wis.).
Example 3
Identification of Anti-ADDL Antibodies
[0137] From the light chain affinity maturation effort, a panel of
seven clones showed strong binding activities to ADDLs when
compared with h3B3 in a phage/Fab ELISA (data not shown). The seven
clones were selected for conversion to IgGs and the monoclonal
antibodies produced and purified for further characterization.
A. Anti-ADDL Antibody Selection
[0138] Following the library panning and screening described in
Example 2, seven leading Fab clones (Tables 3-5) were selected for
IgG conversion. Table 3 shows the amino acid similarity for the
clones selected from the light chain affinity maturation library
relative to parental antibody, h3B3. Table 4A summarizes the number
of amino acid differences in the CDR3 of the light chain of the
selected clones from the CDR3 of the light chain for the parental
antibody, h3B3. Table 4B summarizes the number of amino acid
differences in the CDR1 of the light chain of the selected clones
from the CDR3 of the light chain for the parental antibody, 19.3.
Table 4C summarizes the number of amino acid differences in the
CDR2 of the light chain of the selected clones from the CDR3 of the
light chain for the parental antibody, 19.3. Table 5 is an
alignment of a portion (positions 21-117) of the light chain
variable regions for the selected clones and the parental antibody,
h3B3. CDR3 of each clone is shown in bold.
TABLE-US-00003 TABLE 3 h3B3- humanized Antibody 11.4 17.1 14.2 13.1
19.3 7.2 9.2 LC 11.4 98 98 96 96 96 97 97 17.1 98 96 97 96 97 97
14.2 96 97 98 98 98 13.1 97 97 97 96 19.3 96 97 96 7.2 98 97 9.2
97
TABLE-US-00004 TABLE 4A Number of Amino Acid Differences Antibody
LC-CDR3 sequences from h3B3 h3B3 FQGSHVPPT (SEQ ID NO: 28) 0
(parental) 19.3 FQGSRLGPS (SEQ ID NO: 10) 4 17.1 FQGSRVPAS (SEQ ID
NO: 7) 3 14.2 FQGSRVPPG (SEQ ID NO: 8) 2 13.1 FQGSKAHPS (SEQ ID NO:
9) 4 7.2 FQGSYAPPG (SEQ ID NO: 11) 3 9.2 FQGSRAPPF (SEQ ID NO: 12)
3 11.4 FQGSRVPVR (SEQ ID NO: 13) 3
TABLE-US-00005 TABLE 4B Number of Amino Acid Differences Antibody
LC-CDR1 sequences from 19.3 (parental) 19.3 (parental)
RSSQSIVHSNGNTYLE (SEQ ID NO: 1) 0 19.3 N33S RSSQSIVHSSGNTYLE (SEQ
ID NO: 55) 1 19.3 N33T RSSQSIVHSTGNTYLE (SEQ ID NO: 56) 1 19.3 N33A
RSSQSIVHSAGNTYLE (SEQ ID NO: 57) 1 19.3 N33E RSSQSIVHSEGNTYLE (SEQ
ID NO: 67) 1 19.3 N33D RSSQSIVHSDGNTYLE (SEQ ID NO: 68) 1 19.3
N33S-N35Q RSSQSIVHSSGQTYLE (SEQ ID NO: 59) 2 19.3 N33S-N35S
RSSQSIVHSSGSTYLE (SEQ ID NO: 60) 2 19.3 N33S-N35T RSSQSIVHSSGTTYLE
(SEQ ID NO: 61) 2 19.3 N33S-N35A RSSQSIVHSSGATYLE (SEQ ID NO: 62)
2
TABLE-US-00006 TABLE 4C Number of Amino Acid Differences Antibody
LC-CDR2 sequences from 19.3 (parental) 19.3 (parental) KASNRFS (SEQ
ID NO: 2) 0 19.3 N58Q KASQRFS (SEQ ID NO: 63) 1 19.3 N58S KASSRFS
(SEQ ID NO: 64) 1 19.3 N58T KASTRFS (SEQ ID NO: 65) 1 19.3 N58A
KASARFS (SEQ ID NO: 66) 1
TABLE-US-00007 TABLE 5 17.1
PASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKASNRFSGVPDRFSGSGSGTDFTLKISRVE
AEDVGVYYCFQGSRVPASFGQGTKLEIK (SEQ ID NO: 33) 14.2
PASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKASNRFSGVPDRFSGSGSGTDFTLKISRVE
AEDVGVYYCFQGSRVPPGFGQGTKLEIK (SEQ ID NO: 34) 13.1
PASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKASNRFSGVPDRFSGSGSGTDFTLKISRVE
AEDVGVYYCFQGSKAHPSFGQGTKLEIK (SEQ ID NO: 35) 19.3
PASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKASNRFSGVPDRFSGSGSGTDFTLKISRVE
AEDVGVYYCFQGSRLGPSFGQGTKLEIK (SEQ ID NO: 36) 7.2
PASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKASNRFSGVPDRFSGSGSGTDFTLKISRVE
AEDVGVYYCFQGSYAPPGFGQGTKLEIK (SEQ ID NO: 37) 9.2
PASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKASNRFSGVPDRFSGSGSGTDFTLKISRVE
AEDVGVYYCFQGSRAPPFFGQGTKLEIK (SEQ ID NO: 38) 11.4
PASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKASNRFSGVPDRFSGSGSGTDFTLKISRVE
AEDVGVYYCFQGSRVPVRFGQGTKLEIK (SEQ ID NO: 39) h3B3
PASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKASNRFSGVPDRFSGSGSGTDFTLKISRVE
AEDVGVYYCFQGSHVPPTFGQGTKLEIK (SEQ ID NO: 40)
B. IgG Conversion
[0139] The converted IgGs can be expressed using plasmid based
vectors. The expression vectors were built such that they contain
all the necessary components except the variable regions. In the
basic vectors, the expression of both light and heavy chains was
driven by human CMV promoter and bovine growth hormone
polyadenylation signal. For the seven clones selected for IgG
conversion, the heavy chain variable region was in-frame fused with
a human IgG2 heavy chain constant region (SEQ ID NOS: 20 and 21),
while the light chain variable region was in-frame fused with the
kappa light chain constant region (SEQ ID NOS: 18 and 19). The
heavy (SEQ ID NOS: 29 and 30) and light (SEQ ID NOS: 31 and 32)
chain leader sequences, which mediate the secretion of the
antibodies into the culture media, were also in-frame fused with
the variable regions accordingly. For the heavy chain expression
vectors, the constant region can be selected from a different
subclass isotype, e.g., IgG1 or IgG2. Between the leader sequence
and the constant region, the intergenic sequences contains cloning
sequences for seamless in-frame fusion of the incoming variable
region with the leader sequence at its 5'-end and the constant
region at its 3'-end using In-Fusion cloning strategy (Clontech,
Mountain View, Calif.). The In-Fusion.TM. Dry-Down PCR Cloning Kits
(Clontech, Mountain View, Calif.) was used for PCR amplification of
the variable regions. The dry-down cloning kit contains all the
necessary components for PCR reaction. PCR primers and template
DNAs were added. The expression vectors carry oriP from the EBV
viral genome. The oriP/EBNA1 pair is often used to prolong the
presence of the expression vector inside the transfected cells and
widely used for the extension of the expression duration (Lindner,
et al., 2007, Plasmid 58:1-12) for prolonged expression in 293EBNA
cells, bacterial sequences for a kanamycin selection marker, and a
replication origin in E. coli. When the variable regions were
inserted, the IgGs were directly expressed in mammalian cells. All
heavy chain variable regions herein were cloned into an IgG1
expression vector (pV1JNSA-BF-HCG1) and the light chain variable
regions were cloned into a matching kappa or lambda expression
vector (pV1JNSA-GS-FB-LCK).
C. Antibody Cloning
[0140] The cloning procedure for the resulting antibody expression
vectors was as follows. The variable regions were PCR amplified in
which the PCR reactions were carried out in a volume of 25 .mu.L
containing high fidelity PCR master mix, template volume 1 .mu.L
and forward and reverse primers: 1 .mu.L each. PCR conditions: 1
cycle of 94.degree. C., 2 minutes; 25 cycles of 94.degree. C., 1.5
minutes; 60.degree. C., 1.5 minutes; 72.degree. C., 1.5 minutes and
72.degree. C., 7 minutes; 4.degree. C. until removed. The PCR
products were then digested with DpnI and purified with QIAquick
plate kit (Qiagen, Venlo, The Netherlands). 100 ng of the
corresponding previously linearized heavy chain or light chain
vectors annealed to 10 ng of the PCR fragment with an In-Fusion
reaction (IN-Fusion Dry-Down Cloning Kit, Clontech, Mountain View,
Calif.). The reaction mixture was transformed to XL2 Blue MRF'
competent cells and plated overnight on Agar plates containing 50
.mu.g/mL kanamycin. Light chain constructs were digested with
HindIII+NotI and heavy chain constructs were digested with
AspI+HindIII to check structure by restriction analysis. The DNA
sequences for all the clones were confirmed by sequencing.
D. Antibody Expression in Mammalian Cells and Purification
[0141] Sequencing confirmed constructs of light chain and heavy
chain DNA were transfected in 293 Freestyle cells (Invitrogen,
Carlsbad, Calif.). The 293 Freestyle cells were transfected using
293 Transfectin (Invitrogen, Carlsbad, Calif.). EBNA monolayer
cells were transfected using PEI based transfection reagents.
Transfected cells were incubated at 37.degree. C./5% CO.sub.2 for
seven days in Opti-MEM serum free medium (Invitrogen, Carlsbad,
Calif.). The medium was collected, spun down, filtered through 0.22
um filtration system (Millipore, Billerica, Mass.), and then
concentrated by a Centricon.RTM. centrifuge filter (Millipore,
Billerica, Mass.). Concentrated medium were mixed 1:1 with binding
buffer (Pierce, Thermo Fisher Scientific, Rockford, Ill.), and then
was loaded onto pre-equilibrated protein A/G column (Pierce, Thermo
Fisher Scientific, Rockford, Ill.) or HI trap rProtein A FF from GE
Healthcare, Waukesha, Wis. The loaded column was washed with
binding buffer and eluted with elution buffer (Pierce, Thermo
Fisher Scientific, Rockford, Ill.). Eluted antibody was neutralized
immediately and dialyzed against buffer PBS for overnight. Dialyzed
antibody was concentrated with an Amicon centrifuge filter (Pierce,
Thermo Fisher Scientific, Rockford, Ill.) and protein concentration
was determined by OD.sub.280 nm with the extinct coefficient of
1.34 mg/mL. Purified antibody was analyzed using SDS-PAGE
(Invitrogen, Carlsbad, Calif.), or protein labchip (Caliper
LifeSciences, Hopkinton, Mass.). SDS-PAGE was run under non-reduced
conditions.
[0142] The mutagenesis of the asparagine at position 33 (N33) of
the light chain CDR1 for the antibody 19.3 into N33S (SEQ ID NO:
55), N33T (SEQ ID NO: 56), N33E (SEQ ID NO: 67), or N33D (SEQ ID
NO: 68) was carried out by site directed mutagenesis from the WT
expression vector of pV1JASN-GS-19.3-LCK using QuikChange II XL
Site-Directed Mutagenesis Kit (Agilent Technologies, La Jolla,
Calif.). The codon AAT for N was mutated to AGT for S in 19.3S33
(SEQ ID NO: 55), ACT for T in 19.3T33 (SEQ ID NO: 56), GAA for E in
19.3E33 (SEQ ID NO: 67), or GAT for D in 19.3D33 (SEQ ID NO: 68),
and the new condons in that position were confirmed by DNA
sequencing. To generate full-length IgG antibodies for these
mutants, the respective light chain plasmids were paired with the
cognate heavy chain plasmid, pV1JNSA-19.3-HCG2, for transient
transfection in 293 FreeStyle cells (Invitrogen, Carlsbad, Calif.).
The expression and purification methods were described above in
this example. Aliquots of purified mutant antibodies along with the
19.3 parental antibody (SEQ ID NO: 1) were incubated under various
conditions at 4.degree. C., 25.degree. C. or 40.degree. C. for a
month before being subjected to the ELISA analysis shown in FIGS.
4A-4C.
Example 4
Characterization of Anti-ADDL Antibodies
[0143] The selected anti-ADDL antibodies, i.e. those derived from
the parental antibody, h3B3, where first assessed in a
three-pronged A.beta. ELISA to evaluate binding of the antibody to
monomer A.beta., ADDLs, and fibrillar A.beta.. As shown in FIG. 1,
with the exception of antibody 9.2, all of the anti-ADDL antibodies
showed preferential binding to ADDLs relative to h3B3, selective
(Comp 1 and 3: bind only ADDLs), non-selective (Comp 2: bind all
forms of A.beta. evaluated) comparators, and a control (no
antibody). Antibody 9.2 showed low binding to all forms of A.beta.,
which suggested that its binding affinity was adversely affected
during IgG conversion and/or antibody production. A full titration
curve (FIG. 2) was generated for each antibody and h3B3 to
determine their binding affinity for ADDLs, as compared with
monomer A.beta.. Notwithstanding that six of the seven affinity
matured antibodies showed preferential binding to ADDLs, Applicants
have previously shown that some anti-ADDL antibodies having
preferential binding to ADDLs are not able to prevent ADDL binding
to primary hippocampal neurons (Shughrue, et al., 2010, Neurobiol.
Aging, 31: 189-202, FIG. 1).
[0144] In that preferential binding to ADDLs alone may not be an
accurate predictor of effectiveness, it would be desirable to
identify anti-ADDL antibodies that also block ADDL binding to
neurons, which can be evaluated in a cell-based binding assay as
follows. Antibodies were pre-incubated with ADDLs and then added to
primary hippocampal cultures to assess their blockade of ADDL
binding. The results of this study showed that the anti-ADDL
antibodies herein, specifically antibody 19.3, dramatically reduced
ADDL binding to neurons (FIG. 3). However, a marked reduction in
antibody activity in this assay was observed when the antibodies
were heat-denatured (FIG. 3).
[0145] Determination of EC50. High protein binding plates (Costar,
Corning, Lowell, Mass.), were coated with target ligand in PBS
overnight at 4.degree. C. The concentration of coating protein was
100 pmol/well for A.beta.40 (American Peptide, Sunnyvale, Calif.)
and 50 pmol/well for ADDLs. ADDLs were generated as described in
Example 1B. Next day, plates were washed five times with PBS+0.05%
Tween-20 (Sigma Aldrich, St. Louis, Mo.) and blocked overnight with
Casein blocking buffer (ThermoScientific, Waltham, Mass.) and 0.05%
Tween-20. Three representative antibodies, 19.3 (Fig. A), 19.3S33
(FIG. 4B), and 19.3T33 (FIG. 4C), generated as described in Example
3, were tested at 15 .mu.g/ml to 0 .mu.g/ml in a 12-point
three-fold dilution series. After 2 hours at room temperature
incubation, the plates were washed and alkaline phosphatase
conjugated anti-human IgG (ThermoScientific, Waltham, Mass.) was
added at 0.08 .mu.g/ml. After 45 minutes at room temperature
incubation, the plates were washed and Tropix.RTM. CDP.RTM.-Star
chemiluminescent substrate (Life Technologies.TM., Carlsbad,
Calif.) was added. Luminescence was detected after 30 minutes on an
EnVision.RTM. microplate reader (PerkinElmer, Waltham, Mass.).
Curve fits were completed using GraphPad Prism (GraphPad Software,
Inc., San Diego, Calif.) software.
Example 5
In Vitro FcRn Binding of Anti-ADDL Antibodies
[0146] To characterize the ability of anti-ADDL antibodies to bind
and to dissociate immobilized human FcRn, the seven anti-ADDL
antibodies herein were evaluated in a Biacore FcRn binding assay, a
surrogate system used to evaluate antibody PK and predict the
terminal half life (t.sub.1/2) of antibodies in non-human
primates.
[0147] Briefly, purified human FcRn protein was immobilized onto a
Biacore CM5 biosensor chip and PBSP (50 mM NaPO4, 150 mM NaCl and
0.05% (v/v) Surfactant 20) pH 7.3 was used as running buffer. The
mAbs were diluted with PBSP pH 6.0 to 100 nM, allowed to bind FcRn
for 3 min to reach equilibrium and followed by dissociation in pH
7.3 running buffer. A report point (Stability) was inserted at 5
seconds after the end of mAb binding and the "% bound" was
calculated as RU.sub.Stability/RU.sub.Binding (%). Applicants found
that monoclonal antibodies (mAbs) with identical Fc sequences but
different Fab domains can bind and dissociate from FcRn with
considerable differences (data not shown). Moreover, an apparent
correlation between dissociation at neutral pH and in vivo
pharmacokinetics was observed, in which mAbs with slow-dissociation
fractions (i.e. higher "% bound") tended to exhibit shorter
t.sub.1/2 in vivo.
This feature was used as an in vitro screening tool for antibody
pharmacokinetics.
[0148] A comparison was made of the seven anti-ADDL antibodies
herein, along with h3B3, two ADDL preferring antibodies (Comp 1 and
3) and a non-selective (Comp 2: binds all A.beta. forms evaluated)
comparator in the FcRn binding assay. A sensorgram was generated
(FIG. 5) showing the initial binding of the antibody at pH 6.0 and
then the dissociation of the antibody at pH 7.3 from 180 seconds.
As shown in FIG. 5, there was a noticeable difference between h3B3
and the other antibodies assessed. While h3B3 had a high percent
bound to FcRn, the seven anti-ADDL antibodies of the present
invention, as well as the two comparator antibodies exhibited
considerably lower binding.
Example 6
Characterization of Anti-ADDL Antibody 19.3
[0149] Affinity matured antibody 19.3 was selected for further
characterization. The complete DNA sequence and the deduced amino
acid sequence for the variable region of the light chain was
determined, SEQ ID NOS: 14 and 15, respectively. Alignment of the
heavy (SEQ ID NO: 17) and light (SEQ ID NO: 15) chain variable
regions is shown in FIG. 6A, together with the closest germ line
sequence (SEQ ID NO: 47). A 3D model of heavy and light chain
variable regions and the location of the six complementary
determining regions (CDRs) are shown in FIG. 6B.
[0150] Biacore.TM. (GE Healthcare, Waukesha, Wis.) and KinExA
(Sapidyne, Boise, Id.) analyses were carried out to ascertain the
binding affinity of anti-ADDL antibody 19.3 for ADDLs and determine
the selectivity of 19.3 for ADDLs versus monomer A.beta..
Biacore.TM. and KinExA based technologies are widely used for the
measurement of biding affinity between macromolecules such as
antibody and protein target. In the Surface Plasmon Resonance (SPR)
technology on which Biacore.TM. is based, quantitative measurements
of the binding interaction between one or more molecules are
dependent on the immobilization of a target molecule to the sensor
chip surface. Binding partners to the target can be captured as
they pass over the chip. Surface Plasmon Resonance (SPR) detects
changes in mass in the aqueous layer close to the sensor chip
surface by measuring changes in refractive index. When molecules in
the test solution bind to a target molecule the mass increases
(k.sub.a), when they dissociate the mass falls (k.sub.d). This
simple principle forms the basis of the sensorgram--a continuous,
real-time monitoring of the association and dissociation of the
interacting molecules. The sensorgram provides quantitative
information in real-time on specificity of binding, active
concentration of molecule in a sample, kinetics and affinity.
[0151] The KinExA technology from Sapidyne Instruments, Boise, Id.,
measures binding constants to characterize biomolecular binding
events in the solution phase, not binding events between a solution
phase and a solid phase. In solution, the binding partners reach
equilibrium after sufficient incubation. The unbound molecules are
quantified with a titration, which will reflect the portion of
molecules bound to the partners. The KinExA method does not require
modification of molecules under study. With KinExA, the reaction
being measured occurs between unmodified molecules in solution.
Therefore, concerns of how modification alters "native" binding
reactions are eliminated. The KinExA method allows a wider range of
binding constants as tight as 10.sup.-13 M. The KinExA software
performs data analyses which are based on exact solutions to
classic binding equations (k.sub.d mathematics), not pseudo
first-order approximations. KinExA does not require arbitrary data
manipulations or range selections.
[0152] As shown in Table 6, antibody 19.3 had a 4.8 nM affinity for
ADDLs as compared to a 150 nM affinity for monomer A.beta. in the
Biacore.TM. assay. The thirty fold selectivity of antibody 19.3 for
ADDLs over A.beta. monomer was markedly better than that seen for
the parental antibody, h3B3, which exhibited only a 10 fold
preference for ADDLs versus A.beta. monomer.
TABLE-US-00008 TABLE 6 ADDLs A.beta.1-40 Ratio Antibody (nM) (nM)
(A.beta. monomer/ADDL) 3B3 10.0 104.6 10 19.3 4.8 150.0 31
[0153] Similarly, antibody 19.3 was evaluated in a KinExA based
equilibrium constant measurement. As shown in Table 7, antibody
19.3 had an equilibrium constant of 2.7 nM, which represents more
than a six fold preference for ADDL oligomers versus A.beta.40
monomer binding in the same assay.
TABLE-US-00009 TABLE 7 ADDLs A.beta.1-40 Ratio Antibody (nM) (nM)
(A.beta. monomer/ADDL) 3B3 3.3 45.0 13.6 19.3 2.7 16.7 6.2
Example 7
Biophysical Characterization of Anti-ADDL Antibody 19.3
[0154] Biophysical characterization to assess the potential for
antibody aggregate formation was carrier out to show that the
anti-ADDL antibodies herein are stable under stressed conditions
and suitable for use as a therapeutic. Anti-ADDL antibody 19.3 was
concentrated to >50 mg/mL and placed in a number of formulations
with a pH ranging from 5.0 to 8.0. Two sets of samples were
incubated at 37.degree. C. and 45.degree. C. for one week. A third
set of samples was placed at -70.degree. C. to initiate a series of
five freeze/thaw cycles. Size exclusion chromatography analysis
indicated that the antibody preparations were predominantly
(>95%) in the monomer state, with small amount of dimers, which
were typical for monoclonal antibody preparations. The amount of
dimers and higher molecular weight oligomers did not increase after
the temperature stress across all buffers and no fragmentation was
observed. As summarized in Table 8, the near ultraviolet turbidity
analysis also indicated lack of aggregation. The freeze/thaw
stressed samples showed buffer-dependent increase in turbidity,
which was comparable to other monoclonal antibodies. Viscosity at
50 mg/mL was below 2 centipoise, indicating an acceptable injection
viscosity, as the 20 centipoise level is generally considered to be
a practical limit for subcutaneous injections. Differential
scanning calorimetry also revealed acceptable thermal stability,
with Fab unfolding at about 72.degree. C. and the least stable CH2
domain unfolding above 65.degree. C. Taken together, antibody 19.3
demonstrated very good structural stability with biophysical
properties compatible with subcutaneous delivery.
TABLE-US-00010 TABLE 8 Antibody Initial Aggregates (%) Initial
Fragments (%) 19.3 2.2 0.0 Control 1 1.6 0.4 Control 2 2.6 0.0
Example 8
Pharmacokinetic Analysis of 19.3 and Efficacy in a Model of AD
A. Pharmacokinetics Study in Human FcRn Mice
[0155] Human FcRn mice (heterozygous Tg276) (Jackson Laboratories,
Bar Harbor, Me.) have recently been suggested as a valuable
surrogate system for evaluating monoclonal antibody
pharmacokinetics. To characterize the pharmacokinetics of the
anti-ADDL antibody 19.3 in human FcRn mice, three animals received
a single intravenous injection of antibody 19.3 at 10 mg/kg via
tail vein. A series of 10 .mu.L of blood samples were then
collected at time points 0, 25, 50, 75, 100, 150, 250 and 350 hours
after IV administration of antibody 19.3 or h3B3 and a validated
anti-human IgG immunoassay was used to determine blood levels of
antibody. As shown in FIG. 7, blood levels for antibody 19.3
declined in a biphasic manner with an apparent t.sub.1/2 77.+-.6
hours, which was considerably longer than the half life for the
parental antibody, h3B3, of about 29.+-.9 hours. These half lives
were in agreement with the difference predicted by the in vitro
FcRn binding assay (FIG. 5). The elimination phase terminal half
life was determined using non-compartmental model (WinNonlin.RTM.,
Pharsight, Sunnyvale, Calif.) and data points between day 3 and day
15 post dose.
B. Pharmacokinetics Study in Non-Human Primates
[0156] To confirm the predicted t.sub.1/2 of 19.3 in primates, a
primate pharmacokinetics study was conducted for anti-ADDL antibody
19.3 in a cohort of cisterna magna ported rhesus monkeys. Six
animals (three male/three female) were dosed with a single
intravenous bolus or subcutaneous injection of antibody 19.3 (5
mg/kg) and blood samples collected after antibody administration.
Concurrently, CSF samples were collected from the cisterna magna
port at 0, 2, 4, 8, 12, 24, 30, 48, 54 and 72 hours and the
concentration of antibody 19.3 in the serum and CSF was determined
with an anti-human IgG ELISA assay. When the animals were
administered a single IV bolus injection of antibody 19.3, a
t.sub.1/2 of 254.+-.28 hours was observed, while a t.sub.1/2 of
204.+-.49 hours was seen after subcutaneous administration (FIG.
8). In addition, Applicants found that antibody 19.3 was able to
cross into the primate CSF, where it increased in concentration
during the first 48 hours and peaked at about 0.1% of the antibody
dosed (FIG. 9).
C. Distribution of .sup.125I-Labeled Anti-ADDL Antibody 19.3 in
Mouse Brain
[0157] In an attempt to determine the concentration of antibody
that reached the brain, twelve-month-old male Tg2576 mice (line B6;
SJL-TgN APPSWE) were injected (tail vein) with 200 .mu.g of
.sup.125I-labeled 19.3 antibody (.about.8 mg/kg), or one of two
comparator antibodies, and the blood and CSF collected two hours
later. The residual radioactivity was cleared from the vessels of
the brain via cardiac perfusion with PBS prior to the removal of
the brain. A sample of blood, CSF and the whole brain was then
placed in a gamma counter to determine the amount of radio-labeled
antibody present in each sample. After counting, the brains were
fixed in 4% paraformaldehyde for 48 hours and then processed for
free-floating immunocytochemistry. The localization of antibody
19.3 in the mouse brain was detected with an anti-human secondary
antibody and a standard ABC detection method. This immunoreactivity
was then combined with thioflavin S staining (a stain that detects
plaques) to determine the colocalization of antibody with plaques
in the mouse brain.
[0158] As shown in FIGS. 10A and 10B radiolabeled antibody 19.3 was
able to penetrate the blood-brain-barrier into the mouse CSF and
brain. Moreover, the data indicated that antibody 19.3 was enriched
in the brain (0.19%) when compared with levels seen in the CSF
(0.02%). To determine if this concentration in the brain was due to
the association of antibody 19.3 with A.beta., the brains were
fixed and processed for immunocytochemistry. Analysis of antibody
distribution in the aged Tg2576 mouse brain revealed that antibody
19.3 was associated with thioflavin S positive amyloid plaques in
the brain (FIGS. 10C and 10D). These data provided the first
evidence that antibody 19.3 was able to penetrate into the
transgenic mouse brain and bind to A.beta. species of interest.
Example 9
Plaque Deposition Model
[0159] To further assess the ability of anti-ADDL antibody 19.3 to
abate ADDL deposition into amyloid plaques in the brain,
twelve-month-old male Tg2576 mice (Taconic, N.Y.) were unilaterally
cannulated weekly and bADDLs (50 pmol/.mu.L) infused weekly for
four weeks into the hippocampus (FIG. 11A). One week after the last
bADDL treatment, half of the mice (n=5/treatment) were dosed (tail
vein) weekly, for four weeks with PBS, while the remaining animals
were dosed weekly with 200 .mu.g of anti-ADDL antibody (about 8
mg/kg). All animals were euthanized one week after the last
treatment and their brains processed for immuno-cytochemistry. For
the detection of bADDL and plaques, brain sections were incubated
with Streptavidin Alexa Fluor.RTM. 594 (Invitrogen, Carlsbad,
Calif.), mounted onto slides and the plaques stained with
thioflavin S. Fluorescent images of the plaques were then captured
with a PerkinElmer Rapid Confocal Imager with UltraVIEW ERS
software and the difference in plaque growth quantified. The
details of this model were recently published (Gaspar et. al.,
2010, Exp. Neurol., 223: 394-400). After one month of treatment, a
significant reduction in the deposition of new ADDLs into existing
plaques was seen in animals treated with antibody 19.3 (FIG. 11C),
when compared to animals treated with vehicle alone (FIG. 11B).
Sequence CWU 1
1
68116PRTArtificial Sequencesynthetic 1Arg Ser Ser Gln Ser Ile Val
His Ser Asn Gly Asn Thr Tyr Leu Glu1 5 10 15 27PRTArtificial
Sequencesynthetic 2Lys Ala Ser Asn Arg Phe Ser1 5 39PRTArtificial
Sequencesynthetic 3Phe Gln Gly Ser Xaa Xaa Xaa Xaa Xaa1 5
410PRTArtificial Sequencesynthetic 4Gly Phe Thr Phe Ser Ser Phe Gly
Met His1 5 10 517PRTArtificial Sequencesynthetic 5Tyr Ile Ser Arg
Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val Lys1 5 10 15
Gly68PRTArtificial Sequencesynthetic 6Gly Ile Thr Thr Ala Leu Asp
Tyr1 5 79PRTArtificial Sequencesynthetic 7Phe Gln Gly Ser Arg Val
Pro Ala Ser1 5 89PRTArtificial Sequencesynthetic 8Phe Gln Gly Ser
Arg Val Pro Pro Gly1 5 99PRTArtificial Sequencesynthetic 9Phe Gln
Gly Ser Lys Ala His Pro Ser1 5 109PRTArtificial Sequencesynthetic
10Phe Gln Gly Ser Arg Leu Gly Pro Ser1 5 119PRTArtificial
Sequencesynthetic 11Phe Gln Gly Ser Tyr Ala Pro Pro Gly1 5
129PRTArtificial Sequencesynthetic 12Phe Gln Gly Ser Arg Ala Pro
Pro Phe1 5 139PRTArtificial Sequencesynthetic 13Phe Gln Gly Ser Arg
Val Pro Val Arg1 5 14354DNAArtificial Sequencesynthetic
14gcttctagag atgtggtgat gacccagagc cccctgtccc tgcctgtgac ccctggcgag
60cctgccagca tctcctgccg gagctcccag agcatcgtgc actccaatgg caacacctac
120ctggagtggt acctgcagaa gcctggccag agcccccagc tgctgatcta
caaggcttcc 180aaccggttct ccggcgtgcc tgaccggttc agcggctccg
gcagcggcac agacttcacc 240ctgaagatca gccgggtgga ggctgaggat
gtgggcgtct actactgctt ccagggcagc 300cggcttggtc ctagttttgg
ccagggcacc aagctggaga tcaagcgtac ggtg 35415115PRTArtificial
Sequencesynthetic 15Ala Ser Arg Asp Val Val Met Thr Gln Ser Pro Leu
Ser Leu Pro Val1 5 10 15 Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys
Arg Ser Ser Gln Ser Ile 20 25 30 Val His Ser Asn Gly Asn Thr Tyr
Leu Glu Trp Tyr Leu Gln Lys Pro 35 40 45 Gly Gln Ser Pro Gln Leu
Leu Ile Tyr Lys Ala Ser Asn Arg Phe Ser 50 55 60 Gly Val Pro Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr65 70 75 80 Leu Lys
Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys 85 90 95
Phe Gln Gly Ser Arg Leu Gly Pro Ser Phe Gly Gln Gly Thr Lys Leu 100
105 110 Glu Ile Lys 115 16351DNAArtificial Sequencesynthetic
16gaggtgcagc tggtggagtc cggcggcggc ctggtgcagc ctggcggcag cctgcggctg
60agctgtgctg cctctggctt caccttcagc tcctttggca tgcactgggt gcggcaggcc
120cctggcaagg gcctggagtg ggtggcctac atcagccggg gctccagcac
catctactat 180gctgacacag tgaagggccg gttcaccatc agccgggaca
atgccaagaa ctccctgtat 240ctgcagatga acagcctgcg ggctgaggac
acagcagtgt actactgtgc ccggggcatc 300accacagccc tggactactg
gggccagggc accctggtga ccgtgtccag c 35117117PRTArtificial
Sequencesynthetic 17Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Arg Gly
Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80 Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Gly Ile Thr Thr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu 100
105 110 Val Thr Val Ser Ser 115 18324DNAArtificial
Sequencesynthetic 18cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat
ctgatgagca gttgaaatct 60ggaactgcct ctgttgtgtg cctgctgaat aacttctatc
ccagagaggc caaagtacag 120tggaaggtgg ataacgccct ccaatcgggt
aactcccagg agagtgtcac agagcaggac 180agcaaggaca gcacctacag
cctcagcagc accctgacgc tgagcaaagc agactacgag 240aaacacaaag
tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag
300agcttcaaca ggggagagtg ttag 32419107PRTArtificial
Sequencesynthetic 19Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80 Lys His
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105
20981DNAArtificial Sequencesynthetic 20gcatccacca agggcccatc
cgtcttcccc ctggcgccct gctccaggag cacctccgag 60agcacagccg ccctgggctg
cctggtcaag gactacttcc ccgaaccggt gacggtgtcc 120tggaactctg
gcgccctgac ctctggcgtg cacaccttcc ctgctgtgct gcaatcctct
180ggcctgtact ccctgtcctc tgtggtgaca gtgccatcct ccaacttcgg
cacccagacc 240tacacatgca atgtggacca caagccatcc aacaccaagg
tggacaagac agtggagcgg 300aagtgctgtg tggagtgccc cccatgccct
gccccccctg tggctggccc atctgtgttc 360ctgttccccc ccaagcccaa
ggacaccctg atgatctccc ggacccctga ggtgacctgt 420gtggtggtgg
acgtgtccca tgaggaccct gaggtgcagt tcaactggta tgtggatggc
480gtggaggtgc acaatgccaa gaccaagccc cgggaggagc agttcaactc
caccttccgg 540gtggtgtctg tgctgacagt ggtgcaccag gactggctga
atggcaagga gtacaagtgc 600aaggtgtcca acaagggcct gcctgccccc
atcgagaaga ccatctccaa gaccaagggc 660cagccccggg agccccaggt
gtacaccctg cccccatccc gggaggagat gaccaagaac 720caggtgtccc
tgacctgcct ggtgaagggc ttctacccat ccgacattgc tgtggagtgg
780gagtccaatg gccagcctga gaacaactac aagaccaccc cccccatgct
ggactctgat 840ggctccttct tcctgtactc caagctgaca gtggacaagt
cccggtggca gcagggcaat 900gtgttctcct gctctgtgat gcatgaggcc
ctgcacaacc actacaccca gaagtccctg 960tccctgtccc ctggcaagtg a
98121326PRTArtificial Sequencesynthetic 21Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15 Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50
55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln
Thr65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys
Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro
Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly145 150 155 160 Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175
Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180
185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln
Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu305
310 315 320 Ser Leu Ser Pro Gly Lys 325 2225DNAArtificial
Sequencesynthetic 22tatggcttct agagatgtgg tgatg 252382DNAArtificial
Sequencesynthetic 23tgcagccacc gtacgcttga tctccagctt ggtgccctgg
ccaaaggtgg ggggcacmnn 60mnnmnnmnnm nngcagtagt ag
822470DNAArtificial Sequencesynthetic 24tgcagccacc gtacgcttga
tctccagctt ggtgccctgg ccaaamnnmn nmnnmnnmnn 60gctgccctgg
702524DNAArtificial Sequencesynthetic 25aggcggccct cgaggaggtg cagc
242683DNAArtificial Sequencesynthetic 26agaccgatgg gcccttggtg
gaggcgctgg acacggtcac cagggtgccc tggccccamn 60nmnnmnnmnn mnnggtgatg
ccc 832792DNAArtificial Sequencesynthetic 27agaccgatgg gcccttggtg
gaggcgctgg acacggtcac cagggtgccc tggccccagt 60agtccagmnn mnnmnnmnnm
nnccgggcac ag 92289PRTArtificial Sequencesynthetic 28Phe Gln Gly
Ser His Val Pro Pro Thr1 5 2957DNAArtificial Sequencesynthetic
29atggaatgga gctgggtctt tctcttcttc ctgtcagtaa ctacaggtgt ccactcg
573019PRTArtificial Sequencesynthetic 30Met Glu Trp Ser Trp Val Phe
Leu Phe Phe Leu Ser Val Thr Thr Gly1 5 10 15 Val His
Ser3160DNAArtificial Sequencesynthetic 31atgagtgtgc ccactcaggt
cctggggttg ctgctgctgt ggcttacaga tgccagatgc 603220PRTArtificial
Sequencesynthetic 32Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu
Leu Trp Leu Thr1 5 10 15 Asp Ala Arg Cys 20 3395PRTArtificial
Sequencesynthetic 33Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile
Val His Ser Asn1 5 10 15 Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln
Lys Pro Gly Gln Ser Pro 20 25 30 Gln Leu Leu Ile Tyr Lys Ala Ser
Asn Arg Phe Ser Gly Val Pro Asp 35 40 45 Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser 50 55 60 Arg Val Glu Ala
Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly Ser65 70 75 80 Arg Val
Pro Ala Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 85 90 95
3495PRTArtificial Sequencesynthetic 34Pro Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Ile Val His Ser Asn1 5 10 15 Gly Asn Thr Tyr Leu
Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro 20 25 30 Gln Leu Leu
Ile Tyr Lys Ala Ser Asn Arg Phe Ser Gly Val Pro Asp 35 40 45 Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser 50 55
60 Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly
Ser65 70 75 80 Arg Val Pro Pro Gly Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 85 90 95 3595PRTArtificial Sequencesynthetic 35Pro Ala Ser
Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn1 5 10 15 Gly
Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro 20 25
30 Gln Leu Leu Ile Tyr Lys Ala Ser Asn Arg Phe Ser Gly Val Pro Asp
35 40 45 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
Ile Ser 50 55 60 Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys
Phe Gln Gly Ser65 70 75 80 Lys Ala His Pro Ser Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys 85 90 95 3695PRTArtificial Sequencesynthetic
36Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn1
5 10 15 Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
Pro 20 25 30 Gln Leu Leu Ile Tyr Lys Ala Ser Asn Arg Phe Ser Gly
Val Pro Asp 35 40 45 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile Ser 50 55 60 Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Phe Gln Gly Ser65 70 75 80 Arg Leu Gly Pro Ser Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 85 90 95 3795PRTArtificial
Sequencesynthetic 37Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile
Val His Ser Asn1 5 10 15 Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln
Lys Pro Gly Gln Ser Pro 20 25 30 Gln Leu Leu Ile Tyr Lys Ala Ser
Asn Arg Phe Ser Gly Val Pro Asp 35 40 45 Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser 50 55 60 Arg Val Glu Ala
Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly Ser65 70 75 80 Tyr Ala
Pro Pro Gly Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 85 90 95
3895PRTArtificial Sequencesynthetic 38Pro Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Ile Val His Ser Asn1 5 10 15 Gly Asn Thr Tyr Leu
Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro 20 25 30 Gln Leu Leu
Ile Tyr Lys Ala Ser Asn Arg Phe Ser Gly Val Pro Asp 35 40 45 Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser 50 55
60 Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly
Ser65 70 75 80 Arg Ala Pro Pro Phe Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 85 90 95 3995PRTArtificial Sequencesynthetic 39Pro Ala Ser
Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn1 5 10 15 Gly
Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro 20 25
30 Gln Leu Leu Ile Tyr Lys Ala Ser Asn Arg Phe Ser Gly Val Pro Asp
35 40 45 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
Ile Ser 50 55 60 Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys
Phe Gln Gly Ser65 70 75 80 Arg Val Pro Val Arg Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys 85 90 95 4095PRTArtificial Sequencesynthetic
40Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn1
5 10 15 Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
Pro 20 25 30 Gln Leu Leu Ile Tyr Lys Ala Ser Asn Arg Phe Ser Gly
Val Pro Asp 35 40 45 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile Ser 50 55 60 Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Phe Gln Gly Ser65 70 75 80 His Val Pro Pro Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 85 90 95 41219PRTArtificial
Sequencesynthetic 41Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro
Val Thr Pro Gly1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr
Lys Ala Ser Asn Arg Phe Ser Gly Val Pro 50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65
70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe
Gln Gly 85 90 95 Ser Arg Leu Gly Pro Ser Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150 155 160 Ser Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185
190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
42443PRTArtificial Sequencesynthetic 42Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Gly Ile Thr Thr Ala Leu Asp Tyr Trp
Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr
Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser145 150 155 160 Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn 180
185 190 Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
Asn 195 200 205 Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val
Glu Cys Pro 210 215 220 Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser
Val Phe Leu Phe Pro225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr 245 250 255 Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Gln Phe Asn 260 265 270 Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285 Glu Glu
Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val 290 295 300
Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser305
310 315 320 Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Thr Lys 325 330 335 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu 340 345 350 Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe 355 360 365 Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380 Asn Asn Tyr Lys Thr Thr
Pro Pro Met Leu Asp Ser Asp Gly Ser Phe385 390 395 400 Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 405 410 415 Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425
430 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
43330PRTArtificial Sequencesynthetic 43Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15 Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160 Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180
185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305
310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
44326PRTArtificial Sequencesynthetic 44Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15 Ser Thr Ser Glu Ser
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln
Thr65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys
Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro
Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly145 150 155 160 Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175
Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180
185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln
Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu305
310 315 320 Ser Leu Ser Pro Gly Lys 325 45327PRTArtificial
Sequencesynthetic 45Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Cys Ser Arg1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80 Tyr Thr
Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100
105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
Asn Trp Tyr Val Asp145 150 155 160 Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225
230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg
Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser305 310 315 320 Leu Ser Leu
Ser Leu Gly Lys 325 46326PRTArtificial Sequencesynthetic 46Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20
25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Thr Ser Ser Asn
Phe Gly Thr Gln Thr65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro
Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys
Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly145 150
155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro 195 200 205 Ser Ser Ile Glu Lys Thr Ile Ser Lys
Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn225 230 235 240 Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270
Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275
280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu305 310 315 320 Ser Leu Ser Pro Gly Lys 325
4725PRTArtificial Sequencesynthetic 47Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser 20 25 4814PRTArtificial Sequencesynthetic 48Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser1 5 10
4932PRTArtificial Sequencesynthetic 49Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr Leu Gln1 5 10 15 Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30
5023PRTArtificial Sequencesynthetic 50Asp Ile Val Met Thr Gln Ser
Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15 Glu Pro Ala Ser Ile
Ser Cys 20 5115PRTArtificial Sequencesynthetic 51Trp Tyr Leu Gln
Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr1 5 10 15
5232PRTArtificial Sequencesynthetic 52Gly Val Pro Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr1 5 10 15 Leu Lys Ile Ser Arg
Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys 20 25 30
5316PRTArtificial Sequencesynthetic 53Arg Ser Ser Gln Ser Ile Val
His Ser Xaa Gly Xaa Thr Tyr Leu Glu1 5 10 15 547PRTArtificial
Sequencesynthetic 54Lys Ala Ser Xaa Arg Phe Ser1 5
5516PRTArtificial SequenceSynthetic 55Arg Ser Ser Gln Ser Ile Val
His Ser Ser Gly Asn Thr Tyr Leu Glu1 5 10 15 5616PRTArtificial
SequenceSynthetic 56Arg Ser Ser Gln Ser Ile Val His Ser Thr Gly Asn
Thr Tyr Leu Glu1 5 10 15 5716PRTArtificial SequenceSynthetic 57Arg
Ser Ser Gln Ser Ile Val His Ser Ala Gly Asn Thr Tyr Leu Glu1 5 10
15 5816PRTArtificial SequenceSynthetic 58Arg Ser Ser Gln Ser Ile
Val His Ser Ser Gly His Thr Tyr Leu Glu1 5 10 15 5916PRTArtificial
SequenceSynthetic 59Arg Ser Ser Gln Ser Ile Val His Ser Ser Gly Gln
Thr Tyr Leu Glu1 5 10 15 6016PRTArtificial SequenceSynthetic 60Arg
Ser Ser Gln Ser Ile Val His Ser Ser Gly Ser Thr Tyr Leu Glu1 5 10
15 6116PRTArtificial SequenceSynthetic 61Arg Ser Ser Gln Ser Ile
Val His Ser Ser Gly Thr Thr Tyr Leu Glu1 5 10 15 6216PRTArtificial
SequenceSynthetic 62Arg Ser Ser Gln Ser Ile Val His Ser Ser Gly Ala
Thr Tyr Leu Glu1 5 10 15 637PRTArtificial SequenceSynthetic 63Lys
Ala Ser Gln
Arg Phe Ser1 5 647PRTArtificial SequenceSynthetic 64Lys Ala Ser Ser
Arg Phe Ser1 5 657PRTArtificial SequenceSynthetic 65Lys Ala Ser Thr
Arg Phe Ser1 5 667PRTArtificial SequenceSynthetic 66Lys Ala Ser Ala
Arg Phe Ser1 5 6716PRTArtificial SequenceSynthetic 67Arg Ser Ser
Gln Ser Ile Val His Ser Glu Gly Asn Thr Tyr Leu Glu1 5 10 15
6816PRTArtificial SequenceSynthetic 68Arg Ser Ser Gln Ser Ile Val
His Ser Asp Gly Asn Thr Tyr Leu Glu1 5 10 15
* * * * *